Bacterial strain typing

ABSTRACT

The invention features a method for typing the strain of a bacterial isolate, the method including the steps of: (a) providing genomic DNA from a bacterial isolate; (b) performing a polymerase chain reaction on the genomic DNA using a first and second primer to amplify genomic DNA comprising a restriction nuclease restriction site, thereby producing an amplicon having the restriction site; and (c) characterizing the amplicon of step (b), thereby typing the strain of the bacterial isolate. The invention also features a kit for distinguishing between bacterial strains comprising a set of primer pairs which, when used in a PCR reaction of genomic DNA from a sample of a bacterial isolate amplify DNA across a site for a restriction endonuclease, the amplified DNA being polymorphic between strains of the bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of InternationalApplication No. PCT/US01/44963, filed Nov. 1, 2001, published in Englishunder PCT article 21(2), currently pending, which claims benefit of U.S.provisional application No. 60/244,973, filed Nov. 1, 2000, each ofwhich are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The invention relates to bacterial strain typing.

[0003] In higher plants and animals, the identification of strains orvarieties within a species is a relatively straight forward proposition,since the phenotypic characteristics of the organisms can be examined.However, it is often difficult to appropriately compare differentisolates or strains of bacterial species, since their morphologicalcharacteristics may often be similar or the difference may only beevident in response to specific environmental conditions. Nevertheless,knowledge about the origin, relatedness, and evolution of bacterialspecies is an important area of inquiry, both for epidemiologicalpurposes as well as for the understanding of the evolution andpopulation dynamics of bacterial cultures. This concern becomes ofparticular importance when the genetics of human pathogens isconsidered.

[0004] For example, many outbreaks of infection, particularly those thatare food-borne, now affect patients nearly simultaneously in severaldifferent states or even different countries. Rapid detection of thesewidespread outbreaks may limit spread of disease by allowingidentification and withdrawal of the common source of infection from themarketplace. Development of a rapid, reproducible, and easily comparablestrain typing system for closely related bacterial strains such asenterohemorrhagic E. coli O157:H7 has been a particular challenge. Thisserotype of E. coli emerged as a highly virulent pathogen in the early1980s and has subsequently caused several major outbreaks in the UnitedStates, Europe, and Japan, as well as a large number of sporadicinfections (Kaper and O'Brien, Escherichia coli O157:H7 and other ShigaToxin-Producing E. coli Strains Washington, D.C., ASM Press (1998);Griffin et al., Ann. Intern. Med. 109, 705-712 (1988)). Clinical diseasein humans manifests most commonly as bloody diarrhea (hemorrhagiccolitis), which can progress to the hemolytic-uremic syndrome orthrombotic thrombocytopenic purpura (Griffin and Tauxe, Epidemiol. Rev.13,60-98 (1991)).

[0005] Comparison of two or more isolates of a given bacterial speciesto determine if they are the same or different is a key step in manyepidemiologic, phylogenetic and population studies. Delineation ofisolates of specific human pathogens into distinct related strains, forexample, allows epidemiologists to define outbreaks and to trace thespread of a particular strain in a population (Arbeit, Manual ofClinical Microbiology, ASM Press, pp. 190-208 (1995); Musser, Emerg.Infect. Dis. 2, 1-17 (1996)). Strains of a particular bacterial speciesmay diverge from each other by acquisition or loss of mobile geneticelements, by point mutation, or by other genetic events such asinsertions, deletions, or inversions (Arbeit, Manual of ClinicalMicrobiology, ASM Press, pp. 190-208 (1995)). Some bacterial species,such as Helicobacter pylori, are comprised of highly divergent strainsthat have undergone substantial genetic drift, and even conserved genesin such strains may differ by numerous point mutations (Salau et al.,FEMS Microbiol. Lett. 161, 231-239 (1998)). On the other hand, otherbacteria such as the O157:H7 serotype of Escherichia coli, are highlyclonal, with individual strains containing fewer genetic differences(Wang et al., Nucleic Acids Res. 21, 5930-5933 (1993); Whittam et al.,J. Infect. Dis. 157, 1124-1133 (1988)) (Wang et al., Nucleic Acids Res.21: 5930-5933 (1993); Whittam, Emerg. Infect. Dis. 4: 615-617. (1998)).A number of approaches, both phenotypic and genotypic, have been used toexamine the relatedness of different isolates of a given bacterialspecies or serotype, both for epidemiologic purposes as well as togather insights into the mechanisms of microbial evolution (Musser,Emerg. Infect. Dis. 2, 1-17 (1996); Hill et al., Clin. Microbiol.Newslett. 17, 137-142 (1995)). However, most of these systems for straintyping are limited because of lack of typeability, reproducibility,discriminatory power, ease of interpretation, or ease of performance(Arbeit, Manual of Clinical Microbiology, ASM Press, pp. 190-208(1995)).

[0006] Examples of phenotypic methods for strain typing includebiotyping (carbohydrate fermentation and antimicrobial susceptibilitypattern), serotyping, whole cell fatty acid profiling, phage typing,bacteriocin typing, and multilocus enzyme electrophoresis (MLEt)(Arbeit, Manual of Clinical Microbiology, ASM Press, pp. 190-208 (1995);Steele et al., Appl. Environ. Microbiol. 63, 757-760 (1997)). Of these,MLEE, based on variations in electrophoretic mobilities of enzymesencoded by housekeeping genes, is the most discriminating and has beenused to study the population genetics of different bacterial specieswith reproducible results (Selander et al., Appl. Environ. Microbiol 51,873-884 (1986); Wang et al., Nucleic Acids Res. 21, 5930-5933 (1993);Pupo et al., Infect. Immun. 65: 2685-2692 (1997)). MLEE, however, is alabor intensive and expensive procedure, and may fail to distinguishalleles encoding different enzymes with the same mobility. In addition,MLEE is time-consuming, limiting its applicability in disease outbreaks,where rapidity may help limit spread of the disease (Arbeit, Manual ofClinical Microbiology, ASM Press, 190-208 (1995)). The other phenotypicmethods often suffer from poor discriminative power and/or failure totype all strains (Arbeit, Manual of Clinical Microbiology, ASM Press,pp. 190-208 (1995)).

[0007] Genotypic methods for strain typing have been used increasinglyin recent years. Some of the earlier methods used included restrictionenzyme analysis of plasmid and chromosomal DNA (Arbeit, Manual ofClinical Microbiology, ASM Press, pp. 190-208 (1995)), but spontaneousloss of plasmids and overlapping DNA bands led to confounding patterns,causing these procedures to be replaced with more refined moleculartechniques based on Southern blot hybridization and the polymerase chainreaction (PCR) (Arbeit, Manual of Clinical Microbiology, ASM Press, pp.190-208 (1995); Hill et al., Clin. Microbiol. Newslett. 17, 137-142(1995); Olive and Bean J. Clin. Microbiol. 37, 1661-1669 (1999)).Southern blot hybridization can be used to detect restriction fragmentlength polymorphisms (RFLP) for specific genes, and includes proceduressuch as ribotyping, insertion sequence (IS) typing, and virulence geneprofiling (Arbeit, Manual of Clinical Microbiology, ASM Press, pp.190-208 (1995); Olive and Bean J. Clin. Microbiol. 37, 1661-1669 (1999);Mead and Griffin, Lancet 352, 1207-1212 (1998); Thompson et al., J.Clin. Microbiol. 36, 1180-1184(1998)). Similarly, PCR-based techniques,such as restriction enzyme analysis of PCR products,PCR-based-locus-specific RFLP, repetitive extragenic palindromic elementPCR (Rep-PCR), random amplified polymorphic DNA assay (RAPD), andamplified fragment length polymorphism (AFLP) have all been used forstrain typing (Savelkoul et al., J. Clin. Microbiol. 37, 3083-3091(1999); Wang et al., Nucleic Acids Res. 21, 5930-5933 (1993); Johnsonand O'Bryan Clin. Diagn. Lab. Immunol. 7: 265-273 (2000); Olive andBean, J. Clin. Microbiol. 37, 1661-1669 (1999); Arbeit, Manual ofClinical Microbiology, ASM Press, pp. 190-208 (1995); Mead and Griffin,Lancet 352, 1207-1212 (1998)). Nucleotide sequence analysis andmultilocus sequence typing (MLST) are newer approaches, coupled to therise in genomic sequencing (Olive and Bean, J. Clin. Microbiol. 37,1661-1669 (1999); Feil et al., Mol. Biol. Evol. 16, 1496-1502 (1999);Maiden et al., Proc. Natl. Acad. Sci. U.S.A 95, 3140-3145 (1998)).

[0008] Currently, the molecular technique considered to be the mostreliable and applicable system for strain typing of several bacterialspecies is pulsed-field gel electrophoresis (PFGE) (Tenover et al., J.Clin. Microbiol. 33, 2233-2239 (1995); Olive and Bean, J. Clin.Microbiol. 37, 1661-1669 (1999)). In this procedure, genomic DNA isdigested with a rare cutting restriction endonuclease and PFGE is usedto separate the resulting high molecular size fragments. The distinctiveprofiles generated enable differentiation of strains in a reproduciblemanner. Not all strains, however, are typeable by PFGE. The inability totype certain strains has been ascribed to methylation of restrictionsites, degradation of DNA in agarose plugs, or other technical problems(Johnson et al., Appl. Environ. Microbiol. 61, 2806-2808 (1995); Muraseet al., Curr. Microbiol. 38: 48-50 (1999); Harsono et al., Appl.Environ. Microbiol. 59, 3141-3144 (1993)). While all these moleculartechniques may provide precise data, they are either expensive or timeconsuming to perform, lack sufficient discriminatory power, or requirespecialized equipment. Application of MLST and nucleotide sequenceanalysis techniques to strain typing depends on accurate identificationof polymorphic sites in the genome for comparison (Olive and Bean, J.Clin. Microbiol. 37, 1661-1669 (1999); Feil et al., Mol. Biol. Evol. 16,1496-1502 (1999)). The most important drawback of PFGE is that thecomparison of results for isolates analyzed at different locations ortimes (and hence on different gels) requires sophisticated patternrecognition computer software (Olive and Bean, J. Clin. Microbiol. 37,1661-1669 (1999)). As mentioned above, however, PFGE has certainlimitations as a strain typing system, including time needed foranalysis and the difficulty in comparing patterns of resolved bandsbetween isolates analyzed on different gels. PFGE has also not given anyspecific insights into the mechanisms by which strains of E. coliO157:H7 differ from each other or evolve over time.

[0009] Although several tools are available for strain typing ofbacterial isolates most of these are limited by either lack oftypeability, reproducibility, discriminatory power, ease ofinterpretation, ease of performance, or cost effectiveness, which arethe criteria for evaluating typing systems. Accordingly, a need existsin the art for the development of new approaches to bacterial straintyping.

SUMMARY OF THE INVENTION

[0010] In general, the invention features a method for typing the strainof a bacterial isolate. The method includes the steps of: (a) providinggenomic DNA from a bacterial isolate; (b) performing a polymerase chainreaction on the genomic DNA using a first and second primer to amplifygenomic DNA including a restriction nuclease restriction site, therebyproducing an amplicon having the restriction site; and (c)characterizing the amplicon of step (b), thereby typing the strain ofthe bacterial isolate. In preferred embodiments, the method of theinvention further includes performing a polymerase chain reaction ongenomic DNA of a reference strain of a bacterial isolate using the firstand second primers of step (b) to amplify genomic DNA of the referencestrain of the bacterial isolate, and wherein step (c) is carried out bycharacterizing the amplicon of the reference strain of the bacterialisolate with the amplicon of step (b). In preferred embodiments, thereference strain of the bacterial isolate is E. coli O157:H7 strain86-24. In other preferred embodiments, the method of the inventionfurther includes digesting the amplicon of step (b) with a restrictionnuclease that digests the amplicon at the restriction site and wherestep (c) is carried out by charactering the digestion products.

[0011] In yet other preferred embodiments, the method of the inventionfurther includes performing a polymerase chain reaction on genomic DNAof a reference strain of a bacterial isolate using the first and secondprimers of step (b) to amplify genomic DNA of the reference strain ofthe bacterial isolate and digesting the amplicon of the reference strainwith the restriction nuclease, and where step (c) is carried out bycharacterizing the digestion products of the cleaved amplicon. Onepreferred reference bacterial strain used in the method is E. coliO157:H7 strain 86-24.

[0012] In yet other preferred embodiments, the typing method involvesselecting a restriction site that occurs infrequently in the genome ofthe bacterial isolate. The method also involves the use of a restrictionnuclease such as XbaI or AvrII that cleaves rarely within the genome ofthe bacterial isolate. In still other preferred embodiments, the methodinvolves generating an amplicon of step (b) that includes a PCR fragmenthaving at least 200-400 bp. In other preferred embodiments, the methodinvolves the use of a pathogenic bacterial strain (for example, E. coliO157:H7).

[0013] In still other preferred embodiments, the typing methods involvedetermining whether an amplicon is present in the bacterial isolate thatis not present in the reference strain; an amplicon is absent in thebacterial isolate that is present in the reference strain; or there isan alteration in the size of the amplicon between the bacterial isolateand the reference strain. In other embodiments of the typing method, thedigestion identifies a single nucleotide polymorphism (e.g., identifiesan additional site of restriction nuclease cleavage in the amplicon). Instill other embodiments, the amplicon is digested with at least tworestriction nucleases (e.g., XbaI and AvrII).

[0014] In another aspect, the invention features a method foridentifying a pair of primers for typing a bacterial strain, the methodinvolves the steps of: (a) providing genomic DNA of a bacterial strain;(b) fragmenting the genomic DNA of the bacterial strain into at leasttwo fragments, where the fragments include a restriction enzyme siteflanked by 5′ and 3′ regions of DNA; (c) identifying a first primer thathybridizes to the 5′ region flanking the restriction site and a secondprimer that hybridizes to the 3′ region flanking the restriction site,where the first and second primers amplify genomic DNA of the bacterialstrain having the restriction site; (d) performing a polymerase chainreaction (PCR) on the genomic DNA of the bacterial strain using thefirst and second primers of step (c) to amplify genomic DNA of thebacterial strain, thereby producing an amplicon; (e) providing a secondgenomic DNA, the second genomic DNA being from a reference bacterialstrain; (f) performing a polymerase chain reaction (PCR) on thereference genomic DNA using the first and second primers of step (c) toamplify genomic DNA of the reference bacterial strain, thereby producingan amplicon; (i) comparing the amplicons of step (d) and step (f), wherea difference between the amplicons of steps (d) and (f) identifies thepair of primers as a pair of primers for typing the bacterial strain.

[0015] In preferred embodiments, the method further includes digestingthe amplicons of step (d) and step (f) with a restriction nuclease thatcleaves the amplicons at the restriction site, and further comparing thedigested amplicons of step (d) and (f), wherein a difference between theproducts of the digested amplicons of steps (d) and (f) furtheridentifies the pair of primers for typing the bacterial strain.Exemplary restriction sites useful in the method are those that occurinfrequently in the genome of the bacterial strain. Similarly, arestriction nuclease useful in the method includes enzymes that cleaverarely within the genome of the bacterial strain, for example, XhaI orAvrII.

[0016] In other preferred embodiments, the difference between thebacterial strain and the reference strain is the presence of an ampliconin the bacterial strain that is not present in the reference strain; isthe absence of an amplicon present in the reference strain; or is adifference in the size of the amplicons. In another embodiment, thedigestion identifies a single nucleotide polymorphism (e.g., anadditional site of restriction endonuclease cleavage in the amplicon).In another embodiment, the restriction nuclease is XbaI or AvrII. Inanother embodiment, the amplicon is digested with at least tworestriction nucleases (e.g., XbaI and AvrII).

[0017] In preferred embodiments, the bacterial typing method involves apolymerase chain reaction that amplifies an amplicon of step (c) thatincludes at least 200-400 bp. The method is especially useful foranalyzing pathogen bacterial strains such as E. coli O157:H7. In otherpreferred embodiments, the reference bacterial strain of step (e) is E.coli O157:H7 strain 86-24.

[0018] In other aspects, the invention features a kit for distinguishingbetween bacterial strains. The kit of the invention includes a set ofprimer pairs which, when used in a PCR reaction of genomic DNA from asample of the bacteria amplify DNA across a restriction site for arestriction nuclease, the amplified DNA being polymorphic betweenstrains of the bacteria. In preferred embodiments, the primers areprepared according to the methods disclosed herein.

[0019] In yet another aspect, the invention includes a bacterial straintyping profile, the typing profile produced according to any one of themethods described herein. In preferred embodiments, the typing profileis depicted on an agarose gel or a dot blot or microarray.

[0020] In yet another aspect, the invention features a microarraycomprising at least two amplicons of a pathogenic bacterial strain. Inone embodiment, the microarray contains a collection of amplicons (e.g.,five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety,one hundred, two hundred, three hundred, four hundred, five hundred, orone thousand amplicons, or fragments thereof). In another embodiment,the amplicons, or fragments thereof, are produced as described in any ofthe above aspects. In one preferred embodiment, the pathogenic bacterialstrains are strains of E. coli O157:H7.

[0021] In a related aspect, the invention features a method for typing astrain of a bacterial isolate, the method involves the steps of: (a)providing genomic DNA fragments from a bacterial isolate; (b) detectablylabeling the fragments; (c) contacting the microarray described in theprevious aspect with the detectably labeled fragments; and (d)determining the binding pattern of the fragments to the microarray;thereby typing the strain of the bacterial isolate. In one embodiment,the bacterial strain is a strain of E. coli O157:H7. In anotherembodiment, the isolate is from a patient, a food source, soil, or awater source.

[0022] In a related aspect, the invention features a method of making amicroarray, the method involves the steps of: (a) providing genomic DNAfrom at least one bacterial strain; (b) performing a polymerase chainreaction (PCR) on the genomic DNA of the bacterial strain using a firstand second primer to amplify genomic DNA of the bacterial strain,thereby producing an amplicon; and (c) affixing the amplicon to a solidsupport. In one embodiment, the amplicon is a polymorphic nucleic acidmolecule, or a fragment thereof. In another embodiment, the bacterialstrain is E. coli O157:H7.

[0023] In another aspect, the invention features a method for typing astrain of a bacterial isolate, the method involving the steps of: (a)providing genomic DNA from a bacterial isolate; (b) performing apolymerase chain reaction on the genomic DNA using a first and secondprimer to amplify genomic DNA containing a restriction nucleaserestriction site; and (c) assaying for the presence or absence of theamplicon of step (b), thereby typing the strain of the bacterialisolate.

[0024] The methods disclosed herein provide a straightforward means forstrain typing bacteria and provide numerous advantages over currenttyping systems. For example, the methods of the invention provide aroute for analyzing any number of bacterial isolates recovered fromvirtually any source, including clinical samples and food. The straintyping methods described herein are relatively simple and inexpensive toperform. Moreover, the methods can be performed in any laboratory with athermocycler and other common laboratory materials. In addition, themethods can be performed the very day an isolate is recovered from asample. Interpretation of typing results is also relativelystraightforward as strains are typed on a characteristic profiledetermined by the presence or absence of amplicons. Strain typingresults obtained using the disclosed methods are typically available ina few hours and are highly reproducible.

[0025] By “microarray” is meant an organized collection of at least twonucleic acid molecules affixed to a solid support. Microarrays include,for example, 2, 5, 10, 25, 50, 75, 100, 250, or 500 nucleic acidmolecules.

[0026] By “collection” is meant a group having more than one member. Agroup may be composed of 2, 5, 10, 25, 50, 75, 100, 250, or 500amplicons.

[0027] By “amplicon” is meant a polymorphic nucleic acid molecule, orfragment thereof, produced via a nucleic acid amplification step, suchas a polymerase chain reaction.

[0028] By “polymorphic nucleic acid molecule” is meant a nucleic acidmolecule, or fragment thereof, that is present in one bacterial strain,but that is not present in a reference strain, for example, a referencestrain, such as E. coli O157: H7 strain 933, or 86-24.

[0029] By “fragment” is meant a portion of a nucleic acid molecule(e.g., an amplicon). In some embodiments, the portion is 10, 15, 18, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100,250, 500, 750, or 1000 nucleotides.

[0030] By “typing profile” is meant a reliable representation ofpolymorphic traits that identifies a bacterial strain. For example, amicroarray or dot blot having a characteristic bacterial hybridizationpattern or an agarose gel having a distinctive banding pattern thatidentifies a bacterial strain.

[0031] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1A shows a comparison of pO157 DNA from E. coli O157:H7strain 933, representative isolates G5303 and G5323 and strain 86-24.Identical regions are shown in black and the inserts that differedbetween the strains, in white. The insertions in isolates G5303 andG5323 are identical, but differed from that in strain 86-24. Theinsertion in strain 86-24 contained an XbaI site. Fragment IK8 (ingray), amplified by primer pair IK8A/B, mapped to a region of unknownfunction within pO157 DNA from strain 86-24. This region occurs as a 635bp insertion, relative to this region in strain 933. The sequence at thepoint of insertion is indicated and is identical in all strains shown.

[0033]FIG. 1B shows the original primers (shown in bold) and additionalprimers used for further analysis of the polymorphisms between strains.Primers are in direct alignment with the regions in pO157 DNA fromstrain 86-24 used to design them.

[0034]FIG. 1C shows the agarose gel electrophoresis pattern of ampliconsderived using the primer pairs described in FIG. 1B. The patterngenerated depicts the polymorphism between strains 86-24 and 933diagrammed in FIG. 1A. “M” refers to molecular size marker (100 bp DNAladder; NEB) and “+” or “−” respectively designates the presence orabsence of an amplicon.

[0035]FIG. 2A shows a diagrammatic representation of XbaI-restrictionsite-polymorphisms identified in E. coli O157 strains that areattributable to a substitution-insertion in a lysogenic bacteriophage.Lysogenic phage DNA from E. coli O157:H7 strain 86-24 and strain 933were compared. Identical regions are shown in black and regions thatdiffered between the two strains in white. Strain 933 contains a 2,091bp substitution-insertion containing an XbaI restriction site, betweenthe N and cI genes, in place of a 1,439 bp fragment without an XbaI sitein strain 86-24. Fragment IKB3 (in gray), amplified by the primer pairIKB3A/B, mapped to the substituted region within phage 933W from strain933. Sequence flanking the substitution-insertion is identical betweenthe two strains. Original primers (shown in bold) and additional primersused for further analysis of this polymorphism between strains aredepicted. Primers are in direct alignment with the regions in phage 933Wused to design them.

[0036]FIG. 2B shows a diagrammatic representation of XbaI-restrictionsite-polymorphisms identified in E. coli O157 strains that areattributable to a chromosomal deletion-substitution. Chromosomal DNAsegments from E. coli O157:H7 isolates G5295 and G5296 and strain 933were compared. Identical regions are shown in black and regions thatdiffered between the strains in white. Fragment IK118 (in gray),amplified by primer pair IK118A/B, mapped to a chromosomal region at anO-island-backbone junction in strain 933, and contained an XbaIrestriction site in the O-island sequence. Isolates G5295 and G5296 havea deletion-substitution in this region, substituting a different segmentof DNA at the same location in place of the sequence containing an XbaIrestriction site in strain 933. Original primers (shown in bold) andadditional primers used for further analysis of this polymorphismbetween strains are depicted. Primers are in direct alignment with theregions in the DNA from strain 933 used to design them.

[0037]FIG. 3 is a schematic representation showing a protocol for thedesign of Polymorphic Amplified Typing Sequences (PATS) primer pairs.Genomic DNA fragments derived from E. coli O157: H7 strains 86-24 and933, containing an XbaI restriction site, were selectively cloned intopBluescribe. DNA was initially fragmented using Sau3AI (strain 86-24) orNlaIII (strain 933) restriction enzymes and self-ligated. Thecircularized DNA was then digested with the restriction enzyme XbaI tolinearize only fragments containing an internal XbaI site. Cloning ofthese fragments resulted in plasmids of varying sizes that were prefixedpIK. Insert sequences were determined and used to design PATS primerpairs, shown as divergent block arrows, which flank XbaI restrictionsites in the bacterial genome. “MCS” refers to the multiple cloningsite.

[0038]FIG. 4 shows a representative agarose gel electrophoresis patternof amplicons generated from E. coli O157:H7 isolates using PATS andvirulence gene primer pairs. Presence or absence of amplicons wasisolate specific. Lanes 1-12 show the PCR results of six isolates,obtained using PATS primer pair IK127A/B; the odd number lanes arebefore XbaI digestion and the even lanes, after digestion. Amplicons,when present, always digested with restriction enzyme XbaI into twofragments. Lanes 14-17 show the PCR results of a single isolate (G5299),obtained using virulence gene primer pairs, stx₁F/R, stx₂F/R, eaeF/R,and hlyAF/R. These amplicons lacked an XbaI restriction site and werenot digested with this enzyme (not shown). “M” refers to molecular sizemarker (100 bp DNA ladder; NEB).

[0039]FIGS. 5A and 5B show a phylogenetic analysis of E. coli O157:H7isolates using PATS and PFGE data. Dendrograms were constructed usingthe unweighted pair-group method with arithmetic mean (UPGMA). PFGE gelswere analyzed using Molecular Analyst Fingerprinting Plus software(Bio-Rad) and the data was exported as a band matching table so that thetwo sets of data could be analyzed by the same method. FIG. 5A shows aPATS dendrogram. PATS profiles resolved the isolates into four majorclusters. A genetic distance of <0.1 between each PATS cluster suggestsa clonal lineage for these isolates. The genetic distance is indicatedin increments of 0.01 below the dendrogram. FIG. 5B shows a PFGEdendrogram. PFGE profiles resolved the isolates into smaller clustersand showed greater genetic distance between the isolates.

[0040]FIG. 6 shows the PFGE patterns of the 44 E. coli O157:H7 isolatesfrom 22 outbreaks. Isolate numbers are indicated above the gel. Notethat isolates G5312, G5311, G5306, G5305, G5290, and G5289 could not betyped by PFGE (and are grouped together at the bottom of FIG. 5B). Thelambda DNA ladder standard for PFGE applications (Bio-Rad) was used.Molecular size in kilobase (Kb) is shown to the right.

[0041]FIG. 7 shows multiplex PCR and DNA dot-blot assays to detect PATSpolymorphisms between strains. Target-amplicons were derived from E.coli O157:H7 control strains 86-24 and 933, using each of the eightindicated PATS primer pairs individually. Probe-amplicons were obtainedfrom each of a total of ten isolates, using seven of the eight XbaI PATSprimer pairs in a multiplex PCR reaction and a separate PCR reactionwith primer pair IKB5A/B. These probe-amplicons were hybridized to nylonmembrane strips containing 2.5 μl of each purified target-amplicon. Thehybridization patterns seen on the dot blot autoradiographs matched thecorresponding PATS profiles determined above.

[0042]FIG. 8 shows the DNA sequence (SEQ ID NO: 1) of the O-islandsresiding within the genomic sequence of E. coli O1575:H7 that are notfound in the sequence of the non-pathogenic E. coli strain K12.

[0043] FIGS. 9A-9C show phylogenetic analyses of E. coli O157:H7isolates using PATS data. Dendrograms were constructed using theunweighted pair-group method with arithmetic mean (UPGMA). FIG. 9A showsa dendrogram that was constructed using PATS data from the XbaI primers.FIG. 9B shows a dendrogram that was constructed using PATS data from theAvrII primers. FIG. 9C shows a dendrogram that was constructed bycombining PATS data from the XbaI, AvrII, and virulence gene primers.This approach divided the isolates into smaller clusters showing anincrease in the discriminatory ability of PATS.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention is directed toward a method to efficientlyand accurately type strains of bacteria, particularly pathogenicbacteria. The methodology is based on the discovery that strains ofEscherichia coli O157:H7 differ from each other primarily by insertionsor deletions of nucleic acid sequences and the identification of genomicDNA sequences around each site for a restriction endonuclease which cutsrarely (perhaps 10 to 100 times) within the genome of an organism. PCRamplification of DNA containing the restriction cleavage site is used todetermine the presence, absence, or mutation of the restriction site.Such changes are indicative of genetic variation, and a molecularsubtyping method can be based upon the detection of such geneticvariation.

[0045] At least two approaches are contemplated for deriving theinformation for such a strain typing method. Both methods are intendedto define genomic sequence information centering on the cleavage sitefor the restriction endonuclease.

[0046] In the first approach, small DNA fragments (optimally 200-300base pairs), each containing the restriction cleavage site, are cloned,using a method involving two different restriction endonucleases. Thefragments are created by digesting the whole genomic DNA of the organismwith a restriction endonuclease that cuts the genome many times. Thesmall fragments are then allowed to re-circularize by self-ligation.Then the small fragments are digested using a rare restrictionendonuclease, which cuts and linearizes only the fragments containingthe cleavage site for that endonuclease. The linearized fragments arethen sequenced to determine the sequence of the DNA flanking thecleavage site.

[0047] The second approach is available for those organisms for whichthe whole genomic sequence is available. In that event, a computersearch algorithm can be used to identify all sequences containing thecleavage site as well as the flanking sequences.

[0048] By whichever approach is used, once the cleavage site and theflanking sequence is known, PCR primers are designed to amplify two tofour hundred base pair inserts which would cross over the location ofthe restriction endonuclease cleavage site. Such PCR primers can be usedon genomic DNA of samples of the organism to amplify the DNA of theorganism extending across the cleavage site. Then, if desired, a simpleanalysis of the products of digestion of the PCR products with the rarerestriction endonuclease permits strain typing of the organism.Alternatively, the presence or absence of a PCR product (i.e., anamplicon) is monitored.

[0049] In the first example of the method described herein, forty XbaIrestriction endonuclease sites were identified in strains of Escherichiacoli O157:H7, and forty pairs of primers were designed to amplifygenomic sequences stretching across those sites. A panel of strains ofthe bacterial species was then collected. Genomic DNA from the panel of44 samples of E. coli O157:H7 was isolated, and the primers were used toamplify PCR products containing each of the forty sites for each of thestrains in the panel. A comparison was then done to determine whichprimer pairs were diagnostic of variations between the strains. As itturned out, eight pairs of primers were polymorphic between the strainsand could be used to distinguish strains in the collection from eachother. This exercise demonstrated that it is possible to design arelatively convenient and accurate method of strain typing of bacterialpathogens based on this technique.

[0050] In the next example of the method described herein, primersflanking A vrII sites were designed using the genome sequence of E. coliO157:H7. The primer pairs were used to amplify DNA flanking thirty-threesites in the O157 genome.

[0051] Seven pairs of primers were polymorphic between strains of E.coli O157:H7, and could be used to distinguish the strains. When theseseven polymorphic AvrI primer pairs were used in combination with theeight polymorphic XbaI primer pairs and the primer pairs amplifying fourvirulence genes (stx₁, stx₂, eae, hlyA), the PATS typing systemdistinguished between many more of the bacterial strains than either theXbaI primers or AvrII primers used individually.

[0052] The techniques described above were used specifically to identifya method for typing strains of E. coli O157:H7, a human pathogenicbacteria. As is described in the Examples found below, the rare basecutters XbaI and AvrII were utilized to design a strain typing methodfor E. coli O157:H7.

[0053] In the initial design of the strain typing method for E. coliO157:H7, a collection of 44 strains were collected to be used to testthe primer pairs designed to amplify across the XbaI sites. The fortyprimer pairs were used to create PCR reactions with DNA from each of themembers of the panel. The presence or absence of the PCR products (i.e.,amplicons) was then monitored. It was determined that eight pairs of theprimers produced polymorphic results between the strains of O157:H7 inthe collection. As is discussed below, those primers permittedidentification and typing of the various strains of E. coli O157:H7,both for epidemiological purposes and for the study of the geneticevolution of the pathogen. The sequences of the eight pairs of primersdemonstrated here to be useful for differentiating between strains of E.coli O157:H7 are shown in Table 1A. These eight primer pairs are locatedon larger segments of genomic DNA which are present or absent indifferent stains of E. coli O157:H7. It is contemplated that any primerpairs with these larger genomic regions will work equally well todistinguish amongst the strains.

[0054] In addition, the sequence of the larger genomic regions, referredto as O-islands, since these are islands of DNA sequence that lie withinthe genomic sequence of E. coli O157:H7 that are not found in thesequence of the non-pathogenic E. coli strain K12 are described in FIG.8.

[0055] We also designed primer pairs to amplify DNA flankingthirty-three sites in the O157 genome for another rare cuttingrestriction enzyme, AvrII. Of these sites, we identified seven that werepolymorphic between E. coli O157:H7 isolates. In the case of the AvrIIsites, polymorphisms were due to insertions, deletions, or singlenucleotide polymorphisms (SNPs). The SNPs occurred either within theAvrII site itself, resulting in loss of the site, or in sequences nearthe site, resulting in the creation of an additional AvrII site. Of the7 polymorphic AvrII sites, 5 were in O-islands and 2 were in thebackbone (sequences shared with E. coli K12). Using the primer pairsspecific for DNA flanking these 7 polymorphic AvrII sites with theprimer pairs specific for the 8 polymorphic XbaI sites and the fourvirulence genes (stx₁, stx₂, eae, hlyA), made the bacterial typingsystem described herein highly discriminatory for distinguishing strainsof O157.

[0056] While this method is exemplified in the Examples described hereinwith the strain typing of E. coli O157:H7, it is contemplated that thismethod will work equally well for typing other species or sub-species ofbacteria. Exemplary art-recognized bacteria include, without limitation,foodbome pathogens, non-O157 E. coli, Salmonella species, Listeria (suchas Listeria monocytogenes), Shigella species, Yersinia enterocolitica),Vibrio species, hospital acquired pathogens (such as Enterococcus), andagents of bioterrorism (such as Bacillus anthracis). Other exemplarybacteria include the gram-positive such as Clostridium spp.,Staphylococcus spp., Streptococcus spp. and the gram-negative bacteriasuch as Acinetobacter spp, Bacteroides spp., Bordetella pertussis,Borrelia burgdorferi, Campylobacter spp., Chlamydia trachomatis,Coxiella burnetti, Enterobacter spp., Haemophilus influenzae, Klebsiellaspp., Legionella pneumophila, Mycobacterium spp., Neisseria spp.,Proteus mirabilis, Pseudomonas spp., Xanthomonas spp., and Yersinia spp(such as Yersinia pestis). While the rare base cutter XbaI has beenshown to work well here, it is also contemplated that this method willwork equally well with other restriction endonucleases that cut genomicDNA infrequently. Other such useful art-recognized restriction nucleasesinclude, without limitation, AvrII, SfiI, PacI, NotI, Sse 83871, SrfI,SgrAI, BglII, SpeI, AseI, RsrII, SmaI, SalI, ApaI, CspI, SacII, BlnI,I-Ceul, SwaI, and DpnI. Such restriction enzymes may be used alone or inany combination, for example, according to the methods described herein.

[0057] The following examples are intended to illustrate, not limit, thescope of the invention.

EXAMPLE 1 Strains of Escherichia coli O157:H7 Differ from Each OtherPrimarily by Insertions or Deletions, not by Single NucleotidePolymorphisms

[0058] The recent emergence of Escherichia coli O157:H7 as a humanpathogen may correlate with a hypermutable state and plasticity of theO157 genome. The genetic events related to variations between strains ofE. coli O157:H7 from human outbreaks, which differed from each other bypulsed-field gel electrophoresis patterns following XbaI digestion, wereinvestigated. As is discussed below, this analysis demonstrated thatdifferences between strains of O157:H7 were due to small polymorphicinsertions or deletions containing XbaI sites, rather than to singlenucleotide polymorphisms in the XbaI sites themselves.

[0059] The ability of E. coli O157:H7 to acquire foreign DNA sequencescontributes to the plasticity of its genome (Boerlin, Cell. Mol. LifeSci. 56, 735-741 (1999)). To determine whether the plasticity of theO157 genome is due to hypermutability, a non-biased technique thatdetermines nucleotide sequences flanking each XbaI restriction enzymesite in the O157:H7 genome and compares these sequences betweendifferent strains was performed. The enzyme XbaI was chosen as this ismost commonly used to generate pulsed-field gel electrophoresis (PFGE)typing profiles currently used for differentiating isolates of E. coliO157:H7 (Harsono et al., Appl. Environ. Microbiol. 59, 3141-3144(1993)). The results of this analysis are described below.

[0060] Results

[0061] XbaI Restriction site Polymorphism in E. coli O157 Strains.

[0062] A total of 40 XbaI sites were identified between the genomes ofE. coli O157:H7 reference strains 86-24 and 933. Primer pairs weredesigned that flank each of these 40 XhaI sites and that amplifyapproximately 200-400 bp sized fragments containing these sites. Controlexperiments were set up to test these primer pairs with colony lysatesof strains 86-24 and 933, in a hotstart-touchdown PCR reaction. Thepresence or absence of an amplicon, as well as the presence or absenceof an XbaI site within each amplicon, was assessed by PCR, XbaIdigestion, and agarose gel electrophoresis. The majority of the primerpairs (36 of 40) amplified XbaI-containing DNA fragments of equal sizefrom both strains. However, there were four exceptions: two primer pairsderived from strain 933 failed to yield an amplicon with strain 86-24.Likewise, two primer pairs derived from strain 86-24 did not yieldamplicons when strain 933 DNA was used as the template.

[0063] In addition, these 40 primer pairs were used to analyze 44 E.coli O157:H7 isolates, two isolates each from 22 different outbreakscollected by the Centers for Disease Control and Prevention (CDC).Thirty-two of the 40 primer pairs produced identical results in all 44isolates, with any particular pair generating an amplified product ofidentical size and containing an internal XbaI site. None of the 40primer pairs generated an amplified product that lacked an XbaI site,indicating that none of the 44 O157:H7 isolates contained a singlenucleotide polymorphism or SNP in any of the 40 XbaI sites. On the otherhand, eight primer pairs depicted in Table 1A (below) producedpolymorphic results across the isolate set, amplifying identically sizedproducts with an XbaI site in some isolates but failing to amplify anyproduct in others. TABLE 1 CAPS Cloning/ Tm No. Primer Source LengthSequence (5′→3′) (° C.) 1 IK8A Sau3Al/ 24 GATCTTCTTTTTTAGAGCGCCTTG (SEQID NO:2) 68 IK8B strain 8624 24 TGCCTGAGTTCACAGATAAAACAC (SEQ ID NO:3)68 2 IK25A Sau3Al/ 24 GCGTAATGACTTAATGATTTTCGT (SEQ ID NO:4) 64 IK25Bstrain 8624 24 CATCACATTCCTGACGCAGTGCTT (SEQ ID NO:5) 72 3 IK114ANlaIII/ 24 GAGAATATTATCAGCGACTTGATA (SEQ ID NO:6) 64 IK114B strain 93324 CTAGATCAACTGAGACAGATTATA (SEQ ID NO:7) 64 4 IK118A NlaIII/ 20CATGATTGGCTGGCGTCCCT (SEQ ID NO:8) 64 IK118B strain 933 20ACCAATGAAATGAGTTCAGA (SEQ ID NO:9) 54 5 IK123A NlaIII/ 24TGAAAGTAAACGAAAATTGGCTTC (SEQ ID NO:10) 64 IK123B strain 933 24AAAGAATATCCGGCCCTTCTATCT (SEQ ID NO:11) 68 6 IK127A NlaIII/ 24ATGTTGAGTATATTGGGCAAGACA (SEQ ID NO:12) 66 IK127B strain 933 24GAAATATCGATAACAGACGCTCTC (SEQ ID NO:13) 68 7 IKB3A Strain 933/ 24GAGAAGCCTTGCTTCATTAAAGTA (SEQ ID NO:14) 66 IKB3B Blattner 24ATGAAGCTGTTTTGGCTGCACTAT (SEQ ID NO:15) 68 8 IKB5A Strain 933/ 24ATCTGAAAGATCTGCATTTGATAT (SEQ ID NO:16) 62 IKB5B Blattner 24GATTGTAAGCTAATATCAGCTCAT (SEQ ID NO:17) 64

[0064] In these latter cases, the presence or absence of an amplicon byPCR correlated with the presence or absence of a hybridizing fragment bySouthern blot analysis of genomic DNAs isolated from the correspondingisolates, using control PCR amplicons as probes (data not shown). Asingle exception was observed with one amplicon (IK8) as a probe. Thisfragment hybridized to genomic DNA isolated from all 44 isolates,irrespective of whether an amplified product was obtained from anyparticular isolate using the IK8A/B PCR primer pair. Further evaluationrevealed that one of the IK8 primers (IK8B) corresponded to the 5′ endof the IS629tnp gene, which is widely distributed over the O157 genome(see below).

[0065] The DNA sequences amplified by the 40 primer pairs were analyzedusing the Genbank database (BLAST search program, NCBI) and the E. coliO157:H7 strain 933 genome sequence database (University of Wisconsin).Of the 40 O157:H7 XbaI-containing genome sequences amplified by theprimer pairs, 18 were homologous to E. coli strain K-12 genome sequences(referred to as backbone sequences (Perna et al., Nature 409, 463-466(2001)) and 22 were in regions of the O157:H7 chromosome not shared withK-12, referred to as O-islands (SEQ ID NO.: 1) (Perna et al., Nature409, 463-466 (2001)). The majority of these O-islands (19 of 22)occurred as distinct inserts interrupting homology to the K-12 genome atthe site of insertion. Three of the O-islands replaced other sequencesat the same site on the K-12 genome. All of the eight polymorphicregions that were present in some but not in other E. coli O157:H7isolates were localized to O-islands, compared to 14 of the 32 amplifiedsequences that were conserved across all isolates tested (p<0.01),suggesting that the major genetic differences between O157:H7 strainsoccur in O-island sequences.

[0066] Three of the eight polymorphic regions were analyzed in moredetail to gain insight into the mechanisms underlying straindifferences. Additional primers were designed either from 933 or 86-24genomic sequences to amplify regions upstream, downstream, or across thepolymorphic region being evaluated. The various amplicons were purified,assessed for the presence or absence of an internal XbaI site, andsequenced. This analysis confirmed that all three regions examined,defined by primer pairs IK8A/B, IKB3A/B, and IKI 18A/B, were polymorphicin different O157:H7 isolates because of small insertions or deletionsthat contained XbaI sites, rather than because of single nucleotidepolymorphisms or SNPs in the XbaI sites themselves.

[0067] For example, polymorphism between isolates for theXbaI-containing fragment amplified by IK8A/B was a consequence of asmall insertion in the virulence plasmid. Using the primer pair IK8A/B,an amplicon was obtained from E. coli O157:H7 strain 86-24 but not fromstrain 933. As shown in FIG. 1A, this amplicon, referred to as IK8,specifically extended from a region of unknown function into atransposase gene (IS629tnp) located on the virulence plasmid, pO157, instrain 86-24 (Genbank Accession no. AB011549) (Makino et al., DNA Res.5, 1-9 (1998). The region of unknown function occurred as a 635 bpinsertion in the DNA between the resolvase (redf) and IS629tnp genes instrain 86-24, compared to the sequence of the same region in plasmidpO157 from E. coli O157:H7 strain 933 (FIG. 1A; Genbank Accession no.AF074613) (Burland et al., Nucleic Acids Res. 26, 4196-4204 (1998)); theinsertion in strain 86-24 contained an XbaI site.

[0068] Primer pairs IK8C/D, IK8E/F, and IK8G/H were designed to amplifysections of redF and IS629tnp, and the insertion in strain 86-24 forfurther analysis (FIG. 1B). Identical amplicons were obtained fromstrains 86-24 and 933 using the first two sets of primers, indicatingconservation of the respective genes on both plasmids (FIG. 1C); theseamplicons were not cleaved with XbaI. On the other hand, an amplicon wasobtained with IK8G/H only from strain 86-24 (FIG. 1C) and it containedan XbaI site (data not shown). The primer combination of IK8C/F was usedto amplify the entire length of this region in both strains. The sizedifference in the resulting amplicons (1.2 kb from strain 86-24 and 613bp from strain 933) confirmed the earlier observation that pO157 fromstrain 86-24 contained a 635 bp insertion between bp 850 and 851 ofpO157 in strain 933 (FIG. 1A). BLAST search analysis revealed nohomologies for the inserted sequence in strain 86-24.

[0069] These same primer pairs were used to analyze four additionalisolates of E. coli O157:H7, G5320, G5327, G5303, and G5323, randomlychosen from the CDC isolates that did not yield an amplicon with primerpair IK8A/B. Amplicons derived from isolates G5320 and G5327, usingprimer pair IK8C/F, were of the same size as that from strain 933 (613bp) indicating the absence of an insertion (FIG. 1A). Using theseprimers, amplicons generated from isolates G5303 and G5323 revealed a1.3 kb insert, but this insert did not contain an internal XbaI site(FIG. 1A). Failure to obtain amplicons from isolates G5303 and G5323with primer pairs IK8A/B and IK8G/H showed that isolates G5303 and G5323contained a different insertion than that in 86-24. The sequencesflanking the point of insertion were, however, identical for allisolates tested, including 86-24, G5303, and G5323 (FIG. 1A). BLASTsearch analysis revealed that the insert in isolates G5303 and G5323 had99% homology to three open reading frames (ORFs), L0013, L0014, andL0015, in the LEE pathogenicity island of E. coli O157:H7 strain 933(Perna, N. T. et al. Infect. Immun. 66, 3810-3817 (1998)). These threeORFs comprise ISEc8 in strain 933, an insertion element similar toISRm14 present in Rhizobium and Agrobacterium plasmids (Schneiker etal., Curr. Microbiol. 39, 274-281 (1999)); however, the homologousinsert in isolates G5303 and G5323 contained only part of the L0015 ORFand not the complete IS element. The G+C content was determined for thesequences shared between all isolates (shown as filled-in black arrowsand bars in FIG. 1A; 51%), the inserted sequence in strain 86-24 (33%),and the inserted sequence in strains G5303 and G5323 (55%). The G+Ccontent of E. coli K-12 is 50.8% (Boerlin, Cell. Mol. Life Sci. 56,735-741 (1999); Blattner et al., Science 277, 1453-1474 (1997)). Thelower G+C content of the insert in strain 86-24 is suggestive of apossible heterologous origin (Boerlin, Cell. Mol Life Sci. 56, 735-741(1999); Blattner et al., Science 277, 1453-1474 (1997)). The higher G+Ccontent of the insert in G5303 and G5323 reflects the possible origin ofthis sequence from the Rhizobium and Agrobacterium genomes of high G+C(57 to 63%) composition (Nisslein et al., Appl. Environ. Microbiol. 64,1283-1289 (1998)). These observations suggested that polymorphismsbetween different strains of E. coli O157:H7 reflect the acquisition orloss of small, discrete segments of DNA in the genome, at least some ofwhich may be of heterologous origin.

[0070] Similar analysis of the XbaI-containing fragment amplified byIKB3A/B linked the polymorphism in this region to asubstitution-insertion in a lysogenic bacteriophage. Using the primerpair IKB3A/B, an amplicon was obtained from E. coli O157:H7 strain 933but not from strain 86-24. This amplicon, referred to as IKB3, wasmapped to the lysogenic bacteriophage 933W in strain 933 (GenbankAccession no. AF125520) (Plunkett et al., J. Bacteriol. 181, 1767-1778(1999)). As shown in FIG. 2A, the IKB3 sequence overlapped a 2,091 bpinsertion, containing an internal XbaI site, which was present betweenthe anti-terminator protein (N) and repressor protein (cI) genes inphage 933W. This insertion replaced a 1,439 bp sequence, located atexactly the same site on a similar bacteriophage in E. coli O157:H7strain 86-24, but which lacked an XbaI site (FIG. 2A); hence, thisregion was referred to as a substitution-insertion. Four additionalisolates, G5290, G5325, G5296, and G5301, chosen randomly from the CDCisolates that did not yield an amplicon with primer pair IKB3A/B, wereanalyzed using a primer pair IKB3E/J that would amplify the entirelength of this substitution-insertion (FIG. 2A). No amplicons wereobtained from isolates G5325, G5296, and G5301 (Table 1), indicatingthat this region in these isolates is even more divergent than 86-24from 933. This was confirmed by additional PCR reactions using primerpairs designed to amplify various segments of the region between IKB3Eand IKB3J, which also failed to yield amplicons from the three isolates(data not shown). In contrast, the primer pair IKB3E/J yielded anamplicon from isolate G5290 that was identical in size to that fromstrain 86-24 (Table 1) and lacked an XbaI site. Thus, this region has atleast three variants in the E. coli O157:H7 population studied. TABLE 1Further analysis of the region surrounding the sequence amplified by theprimer pair IKB3A/B. Amplicons derived from E. coli O157 isolates:Primer pairs 86-24 933 G5290 G5325 G5296 G5301 IKB3A/B3B — 193 bp^(a)  — — — — IKB3E/B3J 2.6 kb^(b)  3.2 kb^(a) 2.6 kb^(b) — — —

[0071] Analysis of a third XbaI-containing fragment amplified by IKI18A/B, which also differed between isolates, demonstrated a polymorphismlinked to a deletion-substitution in the chromosome. Using the primerpair IKI 18A/B, an identical amplicon containing an XbaI site wasobtained from most E. coli O157:H7 strains/isolates tested. Thisamplicon, referred to as IK118, was mapped to a chromosomal DNA segmentin E. coli O157:H7 strain 933 that extended across a junction betweenO-island and backbone sequences (FIG. 2B). The backbone sequencecontained the putative transport gene, ypjA (Genbank Accession no.AE000350) (Perna et al., Infect. Immun. 66, 3810-3817 (1998); Rudd,Microbiol. Mol. Biol. Rev. 62, 985-1019 (1998)). While this entireregion, along with its XbaI site, was conserved in most of the E. coliO157:H7 isolates/strains tested, no amplicons were obtained fromisolates G5295 and G5296 using IK 18A/B.

[0072]E. coli O157:H7 strain 933 and isolates G5295 and G5296 wereanalyzed using the primer pair IK118C/D that amplifies across part ofthe O-island and backbone sequence into the 3′ end of ypjA (FIG. 2B). A1.5 kb amplicon containing an XbaI site was obtained from strain 933. Incontrast, isolates G5295 and G5296 had replaced this 1.5 kb region witha different 1 kb of sequence, which lacked an XbaI site, did not containany ORFs, and contained a deletion of the 3′ end of ypjA (FIG. 2B).Hence, this region is referred to as a deletion-substitution. Thedeletion-substitution in G5295 and G5296 may have been caused by theexcision of a prophage in these isolates. Cryptic prophage genes havebeen identified in the O-island region adjacent to thisO-island-backbone junction in E. coli O157:H7 strain 933 (Table 2) (Pemaet al., Nature 409, 463-466 (2001)). TABLE 2 Amplicon Length of derivedfrom associated O- E. coli island in E. coli Position of Xbal O157:H7O157:H7 strain site from one end Description of O- Relation of O-islandto isolates 933 of O-island island E. coli K-12 genome B3 61,664 bp11,088 bp Stx2-encoding Insertion prophage BP-933W 118 21,681 bp 21,637bp Cryptic prophage CP- Replaces unrelated 933Y sequences in K-12 B549,798 bp 36,431 bp Cryptic prophage CP- Partial homology to 933Rcryptic prophage Rac of K-12 114 44,434 bp 8,367 bp Large islandadjacent Replaces unrelated to leuX; includes a sequences in K-12putative site-specific integrase/recombinase, several IS elements,putative helicases and numerous unknowns 123 80,502 bp 35,859 bp Crypticprophage CP- Replaces unrelated 933O sequences in K-12 127 21,120 bp19,318 bp Cryptic prophage CP- Insertion 933T

[0073] In addition to IK8, IKB3, and IK118, the remaining five regionspolymorphic between isolates were also found in O-islands absent in theK-12 genome. Six of the 8 polymorphic regions (IKB3, IK118, IKB5, IK114,IK123, and IK127) were present in strain 933 and the availability of thegenome sequence of this strain allowed us to determine the properties ofthe O-islands containing these six regions (Table 2). The remaining twopolymorphic regions were present in strain 86-24, but not in thesequenced strain 933, the larger genomic context therefore remainedundefined.

[0074] The observations concerning the differences between strains of E.coli O157:H7 are consistent with the conclusion that the high frequencyof mutation among E. coli and Salmonella pathogens is due to theirexistence in a state of transient or permanent hypermutability, whichcan affect both the acquisition of heterologous sequences as well aspoint mutations (LeClerc et al., Science, 274, 1208-1211 (1996)).Specifically, the presence or absence of polymorphic XbaI sites in theO157 genome was found to be a consequence of the insertion or deletionof discrete segments of DNA in the genome, rather than SNPs inindividual XbaI sites. The inserted sequences containing the polymorphicXbaI sites were quite small and usually neither encoded a functionalopen reading frame nor disrupted a pre-existing open reading frame. Anexception was the deletion-substitution observed in isolates G5295 andG5296, which resulted in the loss of 327 bp in the 3′ end of ypjA.However, this deletion did not apparently affect either the viability orpathogenicity of these isolates as they were recovered from humaninfection. The inserted sequences analyzed were not intact insertionsequences, transposons, or bacteriophages. However, several of theinserted sequences were found within O-islands that contained nearbycryptic prophage genes (Table 2), suggesting that phage-mediated eventsmay underlie their acquisition or loss. The inserted sequences wereconsistently found in intergenic regions. Sequences that characterizemutational hot spots or other composition variations (van Belkum et al.,Microbiol. Mol. Biol. Rev. 62, 275-293 (1998)) were not observed in thesequences flanking the insertion points, although each set of insertionsoccurred at exactly the same nucleotide position between strains. Theanalysis of O-islands in the strain 933 genome that contain thesepolymorphic sequences further indicates that the major events drivingevolution of the E. coli O157:H7 genome are not point mutational events,but rather insertions/deletions of discrete DNA sequences.

[0075] Detailed Materials and Methods

[0076] Described below are detailed materials and methods relating tothe above-described experiments. In the case of the XbaI sites, theseexperiments show that strains of Escherichia coli O157:H7 differ fromeach other primarily by insertions or deletions, not by singlenucleotide polymorphisms.

[0077] Bacteria.

[0078]E. coli O157:H7 strain 86-24, streptomycin resistant andoriginally isolated from a human in a Washington State outbreak, waskindly provided by Dr. A. D. O'Brien. E. coli O157:H7 strain 933, ahuman isolate from a Michigan State outbreak, was obtained from theAmerican Type Culture Collection (ATCC, Manassas, Va.) which has itbanked as ATCC 43895. Strain 933 is the O157 isolate that has beensequenced at the University of Wisconsin-Madison, Madison, Wis. Inaddition, 44 isolates of E. coli O157:H7, two each from 22 differentoutbreaks collected by the CDC, Atlanta, Ga., were also included in thisstudy. The isolates from different outbreaks had different PFGE patternssuggesting genetic heterogeneity amongst them. The CDC numbers assignedto these isolates were as follows: G5320, G5327; G5323, G5326; G5321,G5322; G5324, G5325; G5283, G5284; G5285, G5286; G5287, G5288; G5289,G5290; G5291, G5292; G5293, G5294; G5295, G5296; G5297, G5298; G5317,G5318; G5299, G5300; G5301, G5302; G5303, G5304; G5305, G5306; G5307,G5308 (Garden); G5309, G5310 (Meat); G5311, G5312; and G5313, G5314;G5315, G5316. Forty-two of the 44 isolates were isolated from humanclinical cases.

[0079] Design of Primer Pairs Amplifying E. coli O157:H7XbaI Sites.

[0080] Genomic DNA from E. coli O157:H7 strains 86-24 and 933 wasinitially fragmented using Sau3AI (strain 86-24) or NlaIII (strain 933),followed by self-ligation. The circularized DNA was digested with therestriction enzyme XbaI to linearize only fragments containing aninternal XbaI site. These fragments were selectively cloned intopBluescribe (Stratagene USA, LaJolla, Calif.) and sequenced. Insertsequences were used to design twenty-two primer pairs flanking differentXbaI restriction sites; these were prefixed IK. An additional eighteenprimer pairs, with the prefix IKB, were designed using the E. coliO157:H7 strain 933 genomic sequence being assembled at the University ofWisconsin-Madison, Madison. Wis. Additional information on the design ofprimers is provided in Example 2 (below).

[0081] PCR Conditions for Primer Pairs Amplifying XbaI Sites.

[0082] Colony lysates were prepared by boiling colonies suspended insterile distilled water, followed by centrifugation at 4° C. Each E.coli O157:H7 strain template was tested with each individual primer pairin separate reactions. PCR was carried out on the GeneAmp PCR system2400 thermal cycler (PE Biosystems, Foster City, Calif.), using 10 μl ofcolony lysate, 200 pmoles of each primer, 800 μM dNTPs, 1× diluted ExTaq enzyme buffer, and 2.5 units of TaKaRa Ex Taq™ DNA polymerase. Thehot start PCR technique was employed in which the polymerase was addedonly after preheating the rest of the PCR mix (Dieffenbach, C. W. &Dveksler, G. S., eds., PCR Primer—A Laboratory Manual, Cold SpringHarbor Laboratory Press, NY, 1995). This technique was used incombination with a Touchdown PCR profile (Don et al., Nucleic Acids Res.19, 40008 (1991)). To create this profile, the regular PCR program wasmodified as follows: an amplification segment of 20 cycles was set wherethe annealing temperature started at 73° C., to touchdown at 53° C. atthe end of those cycles. Then, another amplification segment of 10cycles was set, using the last annealing temperature of 53° C. Eachreaction was done in triplicate.

[0083] Evaluation of XbaI Amplicons.

[0084] Amplicons obtained by PCR were purified using the QIAQUICK PCRpurification kit and digested with XbaI to confirm the presence of anXbaI site within the amplicon. Undigested and digested DNA fragmentswere resolved on a 4% agarose gel prepared with a combination of 3%Nusieve GTG agarose (FMC BioProducts, Rockland, Me.) and 1% agarose(Shelton Scientific Inc., Shelton, Conn.) and stained with ethidiumbromide. Sequencing of purified amplicons was done at the DNA SequencingCore Facility, Department of Molecular Biology, Massachusetts GeneralHospital. This facility uses ABI Prism DiTerminator cycle sequencingwith AmpliTaq DNA polymerase FS and an ABI 377 DNA sequencer(Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) for thispurpose.

[0085] Southern Blots.

[0086] DNA was fractionated by agarose gel electrophoresis, transferredto Hybond-N+ membranes (Amersham Pharmacia Biotech, Inc., Piscataway,N.J.), U.V. crosslinked to the membrane using a Stratalinker(Stratagene), and hybridized with the appropriate probe, labeled usingthe ECL direct nucleic acid labeling and detection system (AmershamPharmacia). Hybridization at 42° C. and post-hybridization washing ofblots was done as per the ECL kit manual. Autoradiographs were preparedby exposure of processed blots to Kodak Scientific Imaging X-OMAT ARfilm (Eastman Kodak Company, Rochester, N.Y.).

[0087] Data Analysis.

[0088] Statistical analysis was performed using the EpiInfo6 (CDC)software. The significance of differences in proportions was calculatedwith χ² test, or Fisher's exact test if the size of any cell was ≦5. DNA%G+C was determined using the Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis.

Example 2 Polymorphic Amplified Typing Sequences Provide a NovelApproach to Escherichia coli O157:H7 Strain Typing

[0089] As is discussed above, E. coli O157:H7 strains have been shown todiffer from each other by a series of small insertions or deletions ofDNA, some of which contain recognition sites for restriction enzymes.These insertions and deletions determine the complement of XbaIrestriction sites in the genome of a given strain and hence detection ofthese XbaI-containing sequences should provide information comparable toPFGE following XbaI digestion. Below, the potential of directlydetecting these polymorphic sequences by designing a new, simple straintyping system for E. coli O157:H7, which has been termed polymorphicamplified typing sequences or PATS, is demonstrated.

[0090] As is described above in Example 1, using two reference O157strains, a total of forty genomic sequences that contained XhaI siteswere used to generate 40 primer pairs that flanked each individual XbaIsite. These primer pairs were then used to amplify 200-400 bp fragmentsof the surrounding genomic DNAs. In particular, these primer pairs weretested with 44 O157 isolates, two each from 22 different outbreaksinvestigated by the Centers for Disease Control. Of the 40 primer pairs,32 amplified identical XbaI-containing fragments from all 44 isolates,whereas eight produced polymorphic results between isolates, amplifyingidentical XbaI-containing fragments from some but producing no ampliconsfrom others. As is described in more detail below, the 44 isolates weredifferentiated into 14 groups based on which of the eight polymorphicamplicons were detected; phylogenetic analysis divided the isolates intofour major clusters. PATS correctly identified 21 of 22 outbreak pairsas identical or highly related, compared to 14 of 22 identified as suchby PFGE; PATS also was able to type isolates from three outbreaks thatwere untypeable by PFGE. However, PATS was less sensitive than PFGE indiscriminating between outbreaks. These data demonstrated that PATSprovided a simple procedure for strain typing not only O157, but alsoother bacteria.

[0091] Results

[0092] PATS Primer Pairs.

[0093] PATS primer pairs to 40 XbaI sites (and flanking DNA sequences)between the genomes of E. coli O157:H7 strains 86-24 and 933 wereprepared as follows. (A) Using Sau3AI-digested, genomic fragments of E.coli O157:H7 strain 86-24 (FIG. 3): Recombinant plasmids pIK1-100containing E. coli O157:H7 strain 86-24 inserts, derived by digestion ofgenomic DNA by Sau3AI and recovery of inserts containing individual XhaIsites were constructed (FIG. 3). Duplicates among these were eliminatedby Southern blot analysis prior to sequencing (data not shown) andinsert sequences were used to design primer pairs that flanked thegenomic XbaI restriction sites. Of these 100 plasmids, twelve were foundto possess distinct, non-overlapping insert sequences. Primer pairsIK1A/B, IK2A/B, IK8A/B, IK10A/B, IK12A/B, IK18A/B, IK23A/B, IK25A/B,IK38A/B, IK39A/B, IK51A/B, and IK56A/B were derived from these insertsequences. Numbers used to label primer pairs match the pIK plasmid usedto design them.

[0094] (B) Using NlaIII-digested genomic fragments of E. coli O157:H7strain 933 (FIG. 3): Similar to the construction of plasmids pIKI-100,plasmids pIK101-150 contained inserts from E. coli O157:H7 strain 933,derived by digestion of genomic DNA by NlaIII and recovery of insertscontaining individual XbaI sites (FIG. 3). These 50 plasmids wereanalyzed as above and ten of these were found to contain unique insertsequences. Primer pairs IK111A/B, IK114A/B, IK116A/B, IK117A/B,IK118A/B, IK123A/B, IK127A/B, IK131A/B, IK142A/B, and IK148A/B werederived from these insert sequences.

[0095] (C) Using the genome sequence of E. coli O157:H7 strain 933: Ofthe DNA fragments containing XbaI sites identified by sequencing of theE. coli O157:H7 strain 933, 18 did not match sequences alreadyidentified in pIK1-150. Sequences of these 18 fragments were used todesign 18 additional PATS primer pairs designated with IKB numbers(IKB1A/B, IKB3A/B, IKB4A/B, IKB5A/B, IKB6A/B, IKB7A/B, IKB8A/B, IKB9A/B,IKB10A/B, IKB13A/B, IKB14A/B, IKB15A/B, IKB16A/B, IKB17A/B, IKB18A/B,IKB19A/B, IKB20A/B, and IKB21A/B), thereby increasing the overall totalof PATS primer pairs to forty.

[0096] PATS Primer Pairs Amplify Sequences in the E. coli O157:H7 GenomeContaining XbaI Restriction Sites.

[0097] Control PCR experiments were set up to test the PATS primerpairs, using colony lysates and genomic DNA of E. coli O157:H7 strains86-24 and 933 as templates. The PATS primer pairs amplified DNAfragments (one amplicon per primer pair) containing a single XbaIrestriction site, from templates corresponding to the E. coli O157:H7strain used to design them. Identical results were obtained with boththe lysate and purified DNA templates (data not shown).

[0098] The majority of the PATS primer pairs amplified XbaI-containingDNA fragments of identical size from both control strains. However,there were four exceptions. PATS primer pairs IK114A/B and IKB3AIB,derived from strain 933, failed to yield an amplicon with strain 86-24.Likewise, PATS primer pairs IK8A/B and IK25A/B, derived from strain86-24, failed to amplify when strain 933 DNA was used as the template.Thus, the PATS primer pairs were able to establish a discriminatingprofile between the two strains, based on the presence or absence ofamplicons.

[0099] PATS Primers Provide a Strain Typing System for E. coli O157:H7.

[0100] The ability of the 40 PATS primer pairs to discriminate E. coliO157:H7 isolates in a reproducible manner was assessed. To enhance theprofile for each isolate being typed, primer pairs derived from fourvirulence genes (stx₁, stx₂, eae, and hlyA), often (but not always)found in E. coli O157:H7, were also included in the PATS typing system.Based on results obtained with the control strains, colony lysates wereused as templates for PCR and the presence/absence of amplicons, as wellas the presence/absence of an XbaI site within each amplicon, wasassessed by agarose gel electrophoresis. Results were recorded using thedigits 0, 1, or 2, indicating the absence of an amplicon, the presenceof an amplicon without an XhaI site, and the presence of an ampliconwith an XbaI site, respectively.

[0101] Forty-four isolates of E. coli O157:H7, two each from 22different outbreaks (Table 3), were analyzed using this typing system.The presence or absence of an XbaI site within each amplicon wasassessed by agarose gel electrophoresis. A representative agarose gelelectrophoresis pattern of undigested and XbaI-digested ampliconsobtained from some of the isolates is shown in FIG. 4. All ampliconsderived using the PATS primer pairs had a score of 0 or 2; i.e. allisolates that had an amplicon with a given primer pair always had aninternal XbaI site in the amplicon, as seen originally in the controlstrain used to design the PATS primers. Amplicons obtained with thevirulence gene primer pairs had a score 0 or 1. Based on the scoreassigned to each amplicon obtained from every isolate-primer paircombination tested, the 44 E. coli O157:H7 isolates were differentiatedinto 14 PATS types, arbitrarily designated A through N (Table 5). Themost common PATS types were E and G. The reproducibility of this typingsystem was demonstrated by the consistency of profiles obtained in threeseparate analyses of the 44 outbreak isolates. TABLE 3 Summary of E.coli O157:H7 isolates used in this study Outbreak Outbreak IsolatesDescription/Source number location Year 86-24 Human; Sm^(r) strain; Dr.A. D. NA^(a) NA NA O'Brien, personal communication 933 Human; AmericanType Culture NA  NA NA Collection From the Center for Disease Control:G5320, G5327 Human 1 Michigan 1982 G5323, G5326 Human 2 Oregon 1982G5321, G5322 Human 3 Nebraska 1984 G5324, G5325 Human 4 North Carolina1984 G5283, G5284 Human 5 North Carolina 1986 G5285, G5286 Human 6Washington 1986 G5287, G5288 Human 7 Washington 1986 G5289, G5290 Human8 Washington 1986 G5291, G5292 Human 9 Utah 1987 G5293, G5294 Human 10Wisconsin 1988 G5295, G5296 Human 11 Minnesota 1988 G5297, G5298 Human12 Minnesota 1988 G5317, G5318 Human 13 Missouri 1990 G5299, G5300 Human14 Idaho 1990 G5301, G5302 Human 15 Montana 1991 G5303, G5304 Human 16Massachusetts 1991 G5305, G5306 Human 17 Nevada 1992 G5307, G5308 Human,Garden 18 Maine 1992 G5309, G5310 Human, Meat 19 Washington 1993 G5311,G5312 Human 20 Oregon 1993 G5313, G5314 Human 21 Oregon 1993 G5315,G5316 Human 22 Oregon 1993

[0102] The typing patterns observed for these isolates and controlstrains of E. coli O157:H7 were further verified via Southern blotanalysis (data not shown). The presence or absence of an amplicon by PCRcorresponded with the presence or absence of a hybridizing fragment ingenomic DNA, using the control PCR amplicon as a probe (data not shown).A single exception was observed when the IK8A/B amplicon was used as aprobe. This fragment hybridized to DNA from all strains by Southernblot, irrespective of the PCR result. As is described in Example 1, theIK8A/B amplicon partially overlaps the IS629tnp gene, which is widelydistributed over the O157 genome.

[0103] XbaI sites that differ between different O157:H7 strains arelocated on inserted or deleted O157-specific sequences.

[0104] The only differences in the PATS profiles between strainsoccurred with eight of the 40 PATS primer pairs, which amplified regionsof the E. Coli O157:H7 genome that were polymorphic between strains(Tables 4 and 5); that is, these eight primer pairs failed to yield anamplification product in some of the strains tested. These eight PATSprimer pairs included IK8A/B, IK25A/B, IK114A/B, IK118A/B, IK123A/B,IK127A/B, IKB3A/B, and IKB5A/B. Regions amplified by the remaining 32PATS primer pairs were conserved across all strains tested (Tables 4 and5); that is, for each of these 32 primer pairs, all strains tested hadan identically sized PCR product with a conserved XbaI site. As isdescribed in Example 1, the eight PATS primer pairs that yieldedpolymorphic results between strains, amplified regions of DNA that wereinserted or deleted between strains and were all localized in so-calledO-island sequences, which are specific to the 0157 genome and not foundin E. coli K-12 (Table 4). TABLE 4 E. coli O157 genomic regionsamplified by the 40 PATS primer pairs designed flanking XbaI restrictionenzyme sites. Regions conserved across all Regions polymorphic strainsbetween strains Location in E. coli Location in E. coli Primer Pair O157genome Primer Pair O157 genome IK1A/B Backbone^(a) IK8A/B O-islandIK2A/B Backbone IK25A/B O-island IK10A/B Backbone IK114A/B O-islandIK12A/B Backbone IK118A/B O-island IK18A/B O-island^(b) IK123A/BO-island IK23A/B Backbone IK127A/B O-island IK38A/B Backbone IKB3A/BO-island IK39A/B O-island IKB5A/B O-island IK51A/B Backbone IK56A/BBackbone IK111A/B O-island IK116A/B Backbone IK117A/B Backbone IK131A/BBackbone IK142A/B O-island IK148A/B Backbone IKB1A/B O-island IKB4A/BBackbone IKB6A/B O-island IKB7A/B O-island IKB8A/B O-island IKB9A/BO-island IKB10A/B O-island IKB13A/B O-island IKB14A-1/B-1 BackboneIKB15A/B O-island IKB16A/B Backbone IKB17A/B O-island IKB18A/B BackboneIKB19A/B Backbone IKB20A/B O-island IKB21A/B Backbone

[0105] TABLE 5 PATS profiles of E. coli O157:H7 isolates. PCRamplification and XbaI restriction digestion patterns of ampliconsobtained using PATS 40 PATS - 4 virulence gene primer pairs^(b) type^(a)IK1 IK2 IK8 IK10 IK12 IK18 IK23 IK25 IK38 IK39 IK51 IK56 IK111 IK114IK116 IK117 IK118 Control 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 2 Control 2 20 2 2 2 2 0 2 2 2 2 2 2 2 2 2 A 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 2 B 2 20 2 2 2 2 0 2 2 2 2 2 2 2 2 2 C 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 D 2 22 2 2 2 2 0 2 2 2 2 2 2 2 2 2 E 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 F 2 22 2 2 2 2 0 2 2 2 2 2 2 2 2 2 G 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 H 2 22 2 2 2 2 0 2 2 2 2 2 2 2 2 0 I 2 2 0 2 2 2 2 0 2 2 2 2 2 2 2 2 2 J 2 22 2 2 2 2 0 2 2 2 2 2 2 2 2 2 K 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 2 L 2 22 2 2 2 2 0 2 2 2 2 2 2 2 2 2 M 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 N 2 20 2 2 2 2 0 2 2 2 2 2 2 2 2 2 PCR amplification and XbaI restrictiondigestion patterns of amplicons obtained using PATS 40 PATS - 4virulence gene primer pairs^(b) type^(a) IK123 IK127 IK131 IK142 IK148IKB1 IKB3 IKB4 IKB IKB6 IKB7 IKB8 IKB9 IKB10 IKB13 IKB14 Control 2 2 2 22 2 0 2 2 2 2 2 2 2 2 2 Control 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A 2 2 22 2 2 0 2 2 2 2 2 2 2 2 2 B 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 C 2 2 2 2 22 0 2 0 2 2 2 2 2 2 2 D 2 2 2 2 2 2 0 2 0 2 2 2 2 2 2 2 E 2 2 2 2 2 2 22 2 2 2 2 2 2 2 2 F 2 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 G 2 2 2 2 2 2 0 2 22 2 2 2 2 2 2 H 2 2 2 2 2 2 0 2 0 2 2 2 2 2 2 2 I 0 2 2 2 2 2 0 2 2 2 22 2 2 2 2 J 0 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 K 0 2 2 2 2 2 0 2 2 2 2 2 22 2 2 L 2 0 2 2 2 2 0 2 2 2 2 2 2 2 2 2 M 2 2 2 2 2 2 2 2 0 2 2 2 2 2 22 N 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PCR amplification and XbaIrestriction digestion patterns of amplicons obtained using PATS 40PATS - 4 virulence gene primer pairs^(b) type^(a) IKB15 IKB16 IKB17IKB18 IKB19 IKB20 IKB21 stx₁ stx₂ eae hlyA Isolates^(c) Control 2 2 2 22 2 2 0 1 1 1 E. coli O157:H7 strain 86-24 Control 2 2 2 2 2 2 2 1 1 1 1E. coli O157:H7 strain 933 A 2 2 2 2 2 2 2 0 1 1 1 G5289, G5290, G5311,G5312 B 2 2 2 2 2 2 2 1 1 1 1 G5320, G5327 C 2 2 2 2 2 2 2 1 1 1 1G5317, G5324, G5325 D 2 2 2 2 2 2 2 0 1 1 1 G5283, G5284, G5307, G5308 E2 2 2 2 2 2 2 1 1 1 1 G5285, G5286, G5287, G5293, G5294, G5300, G5315,G5321, G5322, G5326 F 2 2 2 2 2 2 2 1 1 1 1 G5288, G5299 G 2 2 2 2 2 2 21 1 1 1 G5291, G5292, G5297, G5298, G5301, G5302, G5309, G5310, G5316 H2 2 2 2 2 2 2 0 1 1 1 G5295, G5296 I 2 2 2 2 2 2 2 1 1 1 1 G5303 J 2 2 22 2 2 2 1 1 1 1 G5304 K 2 2 2 2 2 2 2 1 1 1 1 G5305, G5306 L 2 2 2 2 2 22 1 1 1 1 G5313, G5314 M 2 2 2 2 2 2 2 1 1 1 1 G5318 N 2 2 2 2 2 2 2 1 11 0 G5323

[0106] Phylogenetic Analysis of PATS Profiles Suggests a Clonal Lineagefor E. coli O157:H7 Isolates.

[0107] Based on the PATS profiles, the 44 E. coli O157:H7 isolates weregrouped into four major phylogenetic clusters (FIG. 5A). A geneticdistance of <0.1 between each cluster was suggestive of clonalrelatedness. A closer analysis of the paired isolates from each outbreakwas carried out. The PATS profile type was identical for the twoisolates from 16 of the 22 outbreaks; as an example, isolates G5321 andG5322 belonging to outbreak number 3, shared the PATS profile type E(Tables 3 and 5; FIG. 5A). Isolates from five additional outbreaks(outbreaks 7, 13, 14, 16, and 22) had highly related PATS types, withonly one polymorphism between the paired-isolates; for instance,isolates G5303 and G5304, belonging to outbreak 16, had the PATS profiletypes I and J respectively, differing only by the IK8 fragmentpolymorphism (Tables 3 and 5; FIG. 5A). The remaining two isolates,G5323 and G5326 from outbreak 2, were different due to multiplepolymorphisms (Tables 3 and 5; FIG. 5A); these isolates also hadsubstantially different PFGE patterns (FIG. 6) and so may not, in fact,be related isolates. Overall, the PATS typing system was able tocorrectly relate pairs of isolates from an outbreak for at least 21 ofthe 22 outbreaks (95%) tested (100% if isolates G5323 and G5326 areexcluded). Some isolates from different outbreaks shared a common PATStype, leading to the larger clusters seen in the dendrogram (FIG. 5A),further supporting the clonal descent of these isolates.

[0108] PFGE, the most commonly used current standard for typing E. coliO157:H7, was also used to categorize the 44 isolates from the CDC (FIG.6). The PATS dendrogram was compared with the PFGE dendrogram for theisolates in order to evaluate the potential of these two techniques inrelating/discriminating outbreak-associated E. coli O157:H7.Phylogenetic analysis based on PFGE profiles resolved the 44 CDCisolates into smaller clusters with greater genetic distance betweenthem than PATS. PFGE identified pairs from six outbreaks (outbreaks 3,7, 10, 11, 15, and 16) as identical. For example, isolates G5321 andG5322 from outbreak 3 shared the same PFGE pattern (Table 3, FIGS. 5Band 6). Sixteen isolates from eight outbreaks (outbreaks 4, 5, 9, 12,13, 14, 18, and 21) were classified as probably related (differences of1-3 bands), as defined by Tenover et al (J. Clin. Microbiol. 33,2233-2239 (1995)). For instance, the PFGE patterns of isolates G5317 andG5318, from outbreak 13, differed by one band (Table 3, FIGS. 5B and 6).Ten isolates from five outbreaks (outbreaks 1, 2, 6, 19, and 22) werepossibly related (differences of 4-6 bands). For example, isolates G5320and G5327 from outbreak 1 differed by four bands in the PFGE pattern(Table 3, FIGS. 5B and 6). Six isolates from three outbreaks (outbreaks8, 17, and 20) were untypeable by PFGE (a common problem in PFGE typing)(Table 3, FIGS. 5B and 6). These six isolates were all typeable by PATSand fell into a distinctive cluster (cluster 1 on FIG. 5A).

[0109] PFGE was more discriminatory than PATS, with no overlaps inpatterns between different outbreaks. However, PFGE matched fewer E.coli O157:H7 within outbreaks (pairs from 14 of 22 outbreaks wereclassified as identical or probably related) and was unable to type sixisolates, thereby increasing the complexity of interpretation. Incontrast, PATS typed all 44 isolates and matched 21 of 22 outbreak pairsas identical or related.

[0110] DNA Dot Blots can Effectively Detect PATS Amplicons.

[0111] A dot blot assay to detect PATS amplicons was developed, toassess the feasibility of automating the PATS typing system. Eight PATSprimer pairs that amplified polymorphic regions in the O157 genome wereselected for the assay, as these were critical to the discriminatorypower of PATS (Tables 4 and 5). Using these primer pairs,target-amplicons were derived from E. coli O157:H7 strain 86-24 or 933and were spotted on nylon filters. Multiplex PCR was utilized tosynthesize the probe amplicons to further expedite the assay. Of theeight primer pairs, seven were successfully used in a multiplexreaction. Primer pair IKB5A/B failed to produce sufficient quantities ofits amplicon when used in combination with the other seven primer pairs,irrespective of the template. Altering the primer concentrations,template concentrations, annealing temperatures, extension times, numberof cycles and various additives did not alter the performance ofIKB5A/B. Hence, the probe-amplicon from this primer pair was synthesizedin a separate single primer pair PCR and subsequently purified, labeledand pooled with the rest of the probe-amplicons. Dot blots oftarget-amplicons were hybridized with the probe-amplicons tagged with achemiluminescent label. Resulting hybridization patterns correlatedprecisely with the PATS profiles for the respective isolates (FIG. 7,Table 7).

[0112] This study describes a novel E. coli O157:H7 typing system thatutilizes a technique termed PATS, which is based on the presence orabsence of specific DNA segments in genomic DNA. The technique issimple, highly reproducible and allows accurate objective interpretationof results.

[0113] Typing of pathogenic bacterial strains is important sincedistinct clones within a species/serotype may be associated with diseaseoutbreaks and the severity and frequency of infection (Musser, Emerg.Infect. Dis. 2, 1-17 (1996)). Contemporary molecular typing techniquesin use are based on restriction fragment length polymorphisms ordistribution of random short sequence repeats (Olive and Bean, J. Clin.Microbiol. 37, 1661-1669 (1999); van Belkum et al., Curr. Opin.Microbiol. 2, 306-311(1999)). Of these, PFGE is considered to be the“gold standard” for typing, as it generates distinctive profiles thatdistinguish strains in several serotypes and species, including E. coliO157:H7 (Barrett et al., J. Clin. Microbiol. 32, 3013-3017 (1994); Bohmand Karch, J. Clin. Microbiol. 30, 2169-2172 (1992); Olive and Bean, J.Clin. Microbiol. 37, 1661-1669 (1999)). Since the XbaI restrictionenzyme site occurs infrequently in the O157:H7 genome, it is frequentlyused with PFGE for this organism (Barrett et al., J. Clin. Microbiol.32, 3013-3017 (1994); Bohm and Karch, J. Clin. Microbiol. 30, 2169-2172(1992); Harsono et al., Appl. Environ. Microbiol. 59,3141-3144 (1993)).Although PFGE has been successfully used to support outbreakinvestigations, the technique has its limitations. For example, it maybe impossible to fully resolve all bands on a gel under a single set ofconditions, making interpretation and comparisons difficult (Harsono etal., Appl. Environ. Microbiol. 59,3141-3144 (1993); Johnson et al.,Appl. Environ. Microbiol. 61,2806-2808 (1995); Meng et al., J. Med.Microbiol. 42,258-263 (1995)).

[0114] To overcome problems associated with present typing systems, adifferent typing methodology was developed, which has been termed PATS,based on detecting the presence or absence of the DNA segmentscontaining the polymorphic XbaI sites. PFGE usually resolves about 20-25XbaI-digested fragments for most E. coli O157:H7 isolates (smaller XbaIfragments are not visualized by PFGE) (Barrett et al., J. Clin.Microbiol. 32, 3013-3017 (1994); Harsono et al., Appl. Environ.Microbiol. 59,3141-3144 (1993); Meng et al., J. Med. Microbiol.42,258-263 (1995)). A total of 40 XbaI sites between the genomes of twoE. coli O157:H7 reference strains were identified, and eight of these 40DNA segments were shown to be present or absent across a largecollection of O157 strains. Reproducibility of PATS was demonstrated byconsistency of typing patterns over three repeat PCRs. Compared to PFGE,PATS typed every E. coli O157:H7 isolate tested, matching 21 out of 22outbreak pairs as identical or related and one pair as different. Fourvirulence gene primer pairs into the PATS typing system. Pathogenicityof E. coli O157:H7 is linked to these latter genes and theiridentification would help detect strains with potential for virulence inhumans (Kaper and O'Brien, ASM Press (1998); Paton and Paton J. Clin.Microbiol. 36, 598-602 (1998)). Since the regions amplified by thevirulence gene primer pairs lacked XbaI sites, polymorphisms in thesevirulence genes were distinguished by the presence or absence of theseamplicons.

[0115] In comparison to PATS, PFGE matched fewer E. coli O157:H7 pairswithin outbreaks (pairs from 14 of 22 outbreaks were classified asidentical or highly related) and was unable to type six isolates,thereby increasing the complexity of interpretation. Since the outbreakstrains tested here were collected between 1982 and 1993, it is possiblethat non-matching PFGE patterns of strains from the same outbreak aredue to mutations that occurred during subculturing of the isolates. Itis also possible that some of the isolates were misclassified as beingoutbreak-related, since subtyping was not available at the time of mostof the outbreaks.

[0116] Unlike PFGE, methylation of XbaI sites does not interfere withPATS typing as it is a PCR based procedure (Dieffenbach and Dveksler,Cold Spring Harbor Press, (1995)), thereby avoiding this potentiallyconfounding variable. One drawback of PATS was that it was lessdiscriminatory than PFGE. While PATS detects the presence or absence ofsequences containing XbaI sites, PFGE is also sensitive toinsertions/deletions that may occur between XbaI sites, changing thesize of the intervening fragment without altering the XbaI sitesthemselves. Also, two of the XbaI sites used in the PATS procedure arein DNA segments duplicated elsewhere in the genome (data not shown).While PATS is not dicriminate this duplication (it cannot distinguishbetween one or two copies of identical DNA segments in a genome), suchduplications can affect the PFGE pattern. Although PATS was lessdiscriminatory in our study than PFGE, the precision of the PATSprocedure would be enhanced by identifying additionalinsertions/deletions in O157:H7 isolates and designing correspondingPATS primers.

[0117] PATS is a particularly powerful epidemiological tool for typingE. coli O157:H7 and other bacteria, even when compared to recentlyintroduced typing techniques, such as MLST and octamer-based genomescanning (OBGS) (Kim et al., Proc. Natl. Acad. Sci. U.S.A. 96,13288-13293 (1999)). While MLST can provide unambiguous results that arewidely accessible over websites, the need for sequencing each isolatemay not be cost-effective or provide rapid results (Feil et al., Mol.Biol. Evol. 16, 1496-1502 (1999)). The OBGS technique is similar toenterobacterial repetitive intergenic consensus sequence-PCR (Olive andBean, J. Clin. Microbiol. 37, 1661-1669 (1999)) and has the inherentdisadvantage of relying on repeat sequences; short sequence repeats areapt to undergo variation in composition and position through slippedstrand mispairing during DNA replication and hence, techniques based onthese repeats should be used with caution (van Belkum et al., Microbiol.Mol. Biol. Rev. 62, 275-293 (1998); van Belkum Curr. Opin. Microbiol. 2,306-311 (1999)). Most importantly, as with PFGE, multiple DNA fragmentsgenerated by OBGS require electrophoretic separation and interpretationusing special software (Kim et al., Proc. Natl. Acad. Sci. U.S.A. 96,13288-13293 (1999)).

[0118] Automation according to standard methods would further enhancethe applicability of PATS for routine typing of bacterial isolates. Theconcordance of the results of the DNA dot blot with the results byagarose gel electrophoresis suggests that a variety of techniquesincluding the use of DNA microarrays are useful for such automation ofthe PATS typing system.

[0119] Microarrays

[0120] The present invention provides for nucleic acid compositions thatcan be employed in an array-format for distinguishing between bacterialstrains. These methods are particularly useful for typing bacterialstrains. Microarrays are useful in the diagnosis of a bacterialinfection, in typing the bacterial strain producing the infection, andin determining treatment methods where differing methods of treatmentare indicated by infection with particular bacterial strains.

[0121] The primers of the invention are useful to produce polymorphicnucleic acid fragments that are hybridizable array elements in amicroarray. The array elements are organized in an ordered fashion suchthat each element is present at a specified location on the substrate.Useful substrate materials include membranes, composed of paper, nylonor other materials, filters, chips, glass slides, and other solidsupports. The ordered arrangement of the array elements allowshybridization patterns and intensities to be interpreted as expressionlevels of particular genes. Methods for making nucleic acid microarraysare known to the skilled artisan and are described, for example, in U.S.Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996),and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), hereinincorporated by reference.

[0122] Nucleic Acid Microarrays

[0123] To produce a nucleic acid microarray, primers of the inventionare used to produce amplicons according to the methods described herein.Such amplicons may be synthesized or bound to the surface of a substrateusing a chemical coupling procedure and an ink jet applicationapparatus, as described in PCT application WO95/251116. Alternatively, agridded array may be used to arrange and link amplicon fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedure.

[0124] A nucleic acid molecule (e.g., RNA or DNA) derived from abiological sample (e.g., a bacterial strain infecting a patient) may beused to produce a hybridization probe using standard methods. Thebiological samples are generally derived from a patient, from a bodilyfluid (such as blood, cerebrospinal fluid, phlegm, saliva, urine, orstool) or tissue sample (e.g., a tissue sample obtained by biopsy).Bacterial nucleic acid molecules (RNA or DNA) are isolated according tostandard methods, and a cDNA is produced and used as a template to makecomplementary RNA suitable for hybridization. The RNA is amplified, forexample, in the presence of detectable nucleotides (e.g., fluorescentnucleotides), and the labeled probes are then incubated with themicroarray to allow the probe sequence to hybridize to complementaryoligonucleotides bound to the microarray.

[0125] Incubation conditions are adjusted according to methods known inthe art such that hybridization occurs with precise complementarymatches or with various degrees of less complementarity depending on thedegree of stringency employed. For example, stringent salt concentrationwill ordinarily be less than about 750 mM NaCl and 75 mM trisodiumcitrate, preferably less than about 500 mM NaCl and 50 mM trisodiumcitrate, and most preferably less than about 250 mM NaCl and 25 mMtrisodium citrate. Low stringency hybridization can be obtained in theabsence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least 35% formamide,and most preferably at least 50% formamide. Stringent temperatureconditions will ordinarily include temperatures of at least 30° C., morepreferably of at least 37° C., and most preferably of at least 42° C.Varying additional parameters, such as hybridization time, theconcentration of detergent, e.g., sodium dodecyl sulfate (SDS), and theinclusion or exclusion of carrier DNA, are well known to those skilledin the art. Various levels of stringency are accomplished by combiningthese various conditions as needed. In a preferred embodiment,hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodiumcitrate, and 1% SDS. In a more preferred embodiment, hybridization willoccur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35%formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a mostpreferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl,25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA.Useful variations on these conditions will be readily apparent to thoseskilled in the art.

[0126] The removal of nonhybridized probes may be accomplished, forexample, by washing. The washing steps that follow hybridization canalso vary in stringency. Wash stringency conditions can be defined bysalt concentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least 25° C., more preferably of at least42° C., and most preferably of at least 68° C. In a preferredembodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mMtrisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0127] A detection system may be used to measure the absence, presence,and amount of hybridization for all of the distinct sequencessimultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci.94:2150-2155, 1997). Preferably, a scanner is used to determine thelevels and patterns of fluorescence. The hybridization of bacterialnucleic acid molecules to a particular set of amplicons identifies abacterial strain typing profile.

[0128] Diagnostics

[0129] The hybridization of nucleic acids molecules derived from abacteria is useful in determining the bacterial strain profile. Primers(e.g., those listed in Tables 1A and Table 6), or identified accordingto methods described herein, may be used as targets in a microarray. Themicroarray is used to assay the bacterial strain typing profile.

[0130] In one embodiment, bacteria are isolated from a patient having abacterial infection. Nucleic acid probes derived from the genome ofthese bacteria are hybridized with amplicons, or amplicon fragments,derived from known bacterial strains affixed to a microarray. Thehybridization pattern of the nucleic acid probes defines a particularbacterial strain profile.

[0131] Detailed Materials and Methods

[0132] Described below are detailed materials and methods relating tothe above-described experimental showing that polymorphic amplifiedtyping sequences provide an approach to E. coli O157:H7 strain typing.

[0133] Bacteria, Plasmids and Media Used in this Study.

[0134] (i) E. coli O157:H7: Two strains of E. coli O157:H7 were used inthe standardization of PATS. Strain 86-24, streptomycin resistant andoriginally isolated from a human in a Washington State outbreak, wasobtained from Dr. A. D. O'Brien (Table 3). Strain 933, a human isolatefrom a Michigan State outbreak, was obtained from the American TypeCulture Collection (ATCC, Manassas, Va.) which has it banked as ATCC43895 (Table 3) (Wells et al., J. Clin. Microbiol. 18, 512-520 (1983)).Strain 933 is the E. coli O157:H7 isolate sequenced at the University ofWisconsin-Madison, Madison, Wis. (Perna et al., 2001). In addition, 44isolates of E. coli O157:H7, two each from 22 different outbreaks, wereobtained from the Centers for Disease Control and Prevention (CDC),Atlanta, Ga. The CDC numbers assigned to these isolates and theoutbreaks they were associated with are indicated in Table 3. Theseisolates were primarily human isolates with the exception of two; G5308was isolated from garden manure and G5310 from meat.

[0135] (ii) Other E. coli and plasmids: E. coli DH5α (F⁻ endA1 hsdR17supE44 thi-1 recA1 gyrA96 relA1 Δ(argF-lacZYA)U169 (Φ80d lacZΔM15)) wasused as the host strain to propagate recombinant plasmids. The plasmidpBluescribe (Stratagene USA, LaJolla, Calif.) was used as the cloningvector.

[0136] (iii) Media: All E. coli O157:H7 were grown in Luria-Bertani (LB)media. A single colony from each isolate was used to prepare −80° C.stocks in LB broth with 15% glycerol.

[0137] DNA Extraction, Sequencing, and Probe Labeling.

[0138] Genomic DNA was prepared using the Invitrogen Easy-DNA Isolationkit (Invitrogen Corporation, Carlsbad, Calif.) as per the manufacturer'sinstructions. Plasmid DNA was extracted using Qiagen plasmidpurification kits (Qiagen Inc., Valencia, Calif.). Standardspectrophotometric analysis and agarose gel electrophoresis techniqueswere used to quantitate and evaluate purity of all DNA prepared (Ausubelet al., Current Protocols In Molecular Biology. New York: John Wiley andSons, Inc.(1993); Maniatis, Fritsch, and Sambrook, Molecular cloning: Alaboratory manual. New York: Cold Spring Harbor Laboratory (1989)).

[0139] DNA sequencing was done at the DNA Sequencing Core Facility,Department of Molecular Biology, Massachusetts General Hospital. Thisfacility uses ABI Prism DiTerminator cycle sequencing with AmpliTaq DNApolymerase FS and an ABI 377 DNA sequencer (Perkin-Elmer AppliedBiosystems Division, Foster City, Calif.) for this purpose.

[0140] All DNA probes were labeled using the ECL direct nucleic acidlabeling and detection system (Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.). This approach is based on the direct labeling of DNAprobes with horseradish peroxidase and detection by light generationresulting from the enzymatic cleavage of a chemiluminescent substrate,luminol.

[0141] Identification of Genomic DNA Fragments in E. coli O157:H7Containing an XbaI Restriction Site.

[0142] (i) From Sau3AI-digested genomic DNA of E. coli O157:H7 strain86-24 (FIG. 3): Genomic DNA from strain 86-24 was digested to completionusing the 10 restriction enzyme Sau3AI (New England Biolabs, Inc.,Beverly, Mass.). The digested fragments were allowed to self-ligateovernight and the circularized DNA then digested with XbaI (New EnglandBiolabs); this ensured that only fragments containing an internal XbaIrestriction site would linearize. The linearized fragments were clonedinto the XbaI site in the multiple cloning site of pBluescribe. Theresulting recombinant plasmids are prefixed as pIK. Plasmids wereelectroporated into competent E. coli DH5α cells using standardprotocols (Maniatis et al., Cold Spring Harbor Laboratory (1989)).Transformants were screened on LB plates supplemented with ampicillin(100 μg/ml; Sigma Chemical Co., St. Louis, Mo.),5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 40 μg/ml;Sigma) and isopropyl-β-D-thiogalactopyranoside (IPTG; 1 mM; Sigma). Atotal of 100 white E. coli DH5α colonies containing recombinant plasmidswere selected for further testing. Each strain containing a recombinantplasmid is prefixed IK in this paper.

[0143] (ii) From NlaIII-digested genomic DNA of E. coli O157:H7 strain933 (FIG. 3): A different strain was used to recover NlaIII fragments ofgenomic DNA containing XbaI sites, in order to increase the diversity ofXbaI sites identified, including those not recovered in Sau3AI fragmentsabove. Genomic DNA from strain 933 was digested to completion using therestriction enzyme NlaIII (New England Biolabs). Subsequent stepsleading to the selection of XbaI-containing fragments and the finalscreening of recombinant clones were as above. A total of 50 white E.coli DH5α colonies containing recombinant plasmids were selected forfurther testing. Plasmids and colonies were named as indicated above.

[0144] (iii) From E. coli O157:H7 strain 933 genomic DNA sequence: Atotal of 40 XhaI sites were localized in the 933 genomic sequenceassembled at the University of Wisconsin-Madison, Madison, Wis., ofwhich two were in duplicated regions and were not included in thisstudy. Of the remaining 38 XhaI sites, 20 were already identified inplasmids described above, and 18 were newly identified from the genomesequence. The sequences surrounding these 18 XbaI sites are referred towith the prefix IKB in this paper. Two additional XbaI-containinggenomic segments are unique to strain 86-24 and were recovered in step(i) above.

[0145] Evaluation of Recombinant Plasmids.

[0146] Plasmid DNA was extracted from isolated colonies of IK1-150 andplasmids pIK1-150 were screened for the presence of an appropriateinsert. As a result of the self-ligation at the Sau3AI or NlaIII sites,digestion with XbaI and cloning, an appropriate insert would have XbaIsites at either end, and a single, internal Sau3AI or NlaIII site (FIG.3). Plasmids were digested with XbaI to check for the release of asingle insert. In addition, pBluescribe-specific primers (see below)were used to amplify the insert by PCR. The resulting amplicons werepurified using the Qiaquick PCR purification kit (Qiagen, Inc.) and thendigested with either Sau3AI or NlaIII, to confirm the presence of thesesites within the fragments. DNA fragments were resolved by agarose gelelectrophoresis and visualized by staining with ethidium bromide. ThepBluescribe-specific primers were in the multiple cloning site on eitherside of the insert, and were: Reverse (5′-GAAACAGCTATGACC ATG-3′; SEQ IDNO.: 18) and M13-20 (5′-GTAAAACGACGGCCAGT-3′; SEQ ID NO:19). PCR wasdone on a PTC-100 thermal cycler (MJ Research, Inc., Watertown, Mass.),using 10 ng plasmid DNA, 100 pmoles of each vector primer, 800 μM dNTPs,1× diluted Ex Taq™ enzyme buffer and 2.5 units of TaKaRa Ex Taq™ DNApolymerase (Takara Shuzo Co., LTD., Panvera Corporation, Madison, Wis.).Denaturation at 95° C. for 5 min was followed by 30 cycles ofamplification (1 min at 95° C., 1 min at 45° C., 1 min at 72° C. percycle) and a final extension at 72° C. for 1 min. Each reaction was donein triplicate.

[0147] As more recombinant plasmids were studied, duplicates containinginserts already analyzed were eliminated using Southern blothybridization. Briefly, XbaI-digested plasmid DNA was electrophoresed onan agarose gel, transferred to Hybond-N+ membranes (Amersham Pharmacia),U.V. crosslinked to the membrane using a Stratalinker (Stratagene), andhybridized with a pool of the previously characterized insert DNAslabeled as described above. Hybridization at 42° C. andpost-hybridization washing of blots was done as per the ECL kit manual(Amersham Pharmacia). Autoradiographs were prepared by exposure ofprocessed blots to the Kodak Scientific Imaging X-OMAT AR film (EastmanKodak Company, Rochester, N.Y.), and plasmids containing insertshybridizing to the pool of previous inserts were not further evaluated.

[0148] Design of PATS and Virulence Gene Primer Pairs.

[0149] Plasmids with appropriate inserts were sequenced usingpBluescribe specific primers (reverse and M13-20). Insert sequences wereused to design PATS primer pairs flanking each XbaI site on the genomeand designed to amplify fragments of approximately 200-400 bp (FIG. 3).In the context of the plasmid, these primers appear divergent to eachother, since the genomic XbaI site is linearized at either end of theinsert (FIG. 3). However, in the undigested genome, each primer pairflanks a single, internal XbaI site. PATS primer pairs were alsodesigned to amplify the 18 XbaI sites specifically identified from theE. coli O157:H7 strain 933 genome sequence.

[0150] Primer pairs were also generated to amplify specific virulencegenes found in strains of E. coli O157:H7, similar to those designed byPaton et al (Paton and Paton, J. Clin. Microbiol. 36, 598-602 (1998)).The four primer pairs included: stx₁F (5′-ATAAATCGCCATTCGTTGACTAC-3′;SEQ ID NO:20)/ stx₁R (5′-GAACGCCCACTGAGATCAT C-3′; SEQ ID NO:21), stx₂F(5′-GGCACTGTCTGAAACTGCTCC-3′; SEQ ID NO:22)/ stx₂R(5′-TCGCCAGTTATCTGACAT TCTG-3′; SEQ ID NO:23), eaeF(5′-GACCCGGCACAAGCATA AGC-3′; SEQ ID NO:24)/ eaeR (5′-CCACCTGCAGCAACAAGAGG-3′; SEQ ID NO:25) and hlyAF (5′-GCATCATCAAGCGT ACGTTCC-3′; SEQID NO:26)/ hlyAR (5′-AATGAGCCAAGCTGGTTAAGCT-3′; SEQ ID NO:27).

[0151] PATS Typing.

[0152] PATS primers were used to assay for the presence or absence ofindividual XbaI sites in different isolates of E. coli O157:H7. PCR wasdone using E. coli O157:H7 colony lysate and/or genomic DNA astemplates. Colony lysates were prepared by boiling a suspension ofcolonies in sterile distilled water, followed by centrifugation at 4° C.Each E. coli O157:H7 isolate template was tested with each individualPATS primer pair, in separate reactions.

[0153] PCR was done on the GeneAmp PCR system 2400 thermal cycler (PEBiosystems, Foster City, Calif.), using 200 ng genomic DNA or 10 μl ofcolony lysate, 200 pmoles of each PATS primer, 800 μM dNTPs, 1× dilutedEx Taq™ enzyme buffer and 2.5 units of TaKaRa Ex Taq™ DNA polymerase.Hot start PCR technique was employed in which the polymerase was addedonly after preheating the rest of the PCR mix (Dieffenbach and Dveksler,Cold Spring Harbor Press (1995)). This technique was used in combinationwith a Touchdown PCR profile (Lawrence and Hartl, Genetica 84, 23-29(1991)). To create this profile, the regular PCR program was modified asfollows: an amplification segment of 20 cycles was set where theannealing temperature started at 73° C., to touchdown at 53° C. at theend of those cycles. Then, another amplification segment of 10 cycleswas set, using the last annealing temperature of 53° C. Each reactionwas done in triplicate.

[0154] Amplicons obtained by PCR were purified using the Qiaquick PCRpurification kit and digested with XbaI to confirm the presence of anXbaI site within the amplicon. Undigested and digested DNA fragmentswere resolved on a 4% agarose gel prepared with a combination of 3%Nusieve GTG agarose (FMC BioProducts, Rockland, Me.) and 1% agarose(Shelton Scientific Inc., Shelton, Conn.), stained with ethidiumbromide. These same amplicons were also used to probe genomic DNA ofisolates used in PATS typing, following digestion with Sau3AI.

[0155] Pulsed-Field Gel Electrophoresis (PFGE).

[0156] PFGE analysis of all E. coli O157:H7 isolates was done at theCDC, Atlanta, Ga. Standard procedures previously described (Ausubel etal., Current Proocols in Moelcular Biology, John Wiley and Sons, Inc.(1993); Barrett et al., J. Clin. Microbiol. 32, 3013-3017 (1994)) wereused, with the following modifications. Briefly, genomic DNA of eachisolate was embedded in separate agarose plugs and digested at 37° C.for 2 hr with 30U of XbaI per plug (Gibco BRL, Grand Island, N.Y.). Theplugs were loaded onto a 1% agarose-Tris buffer gel (SeaKem GoldAgarose, BioWhittaker Molecular Applications, Rockland, Mass.) and PFGEwas performed using a CHEF Mapper XA (Bio-Rad Laboratories, Hercules,Calif.). DNA was electrophoresed for 18 h at a constant voltage of 200 V(6 V/cm), with a pulse time of 2.2 to 54.2 s, an electric field angle of120°, and temperature of 14° C., before being stained with ethidiumbromide.

[0157] DNA Dot-Blots.

[0158] Primer pairs IK8A/B, IK25A/B, IK114A/B, IK118A/B, IK123A/B,IK127A/B, IKB3A/B, and IKB5A/B were used in this assay. Amplicons werefirst obtained from E. coli O157:H7 strain 86-24 or 933, using eachprimer pair in a separate reaction. 2.5 μl of each purified amplicon wasspotted on Hybond N+ membrane (Amersham Pharmacia) strips and U.V.crosslinked; these constituted the “target-amplicons”. Ten E. coliO157:H7 isolates (G5301, G5302, G5295, G5296, G5323, G5326, G5313,G5314, G5303, and G5304), from five different outbreaks, were selectedfor analysis by dot-blot using multiplex PCR. For each of theseisolates, amplicons were derived using seven of the eight primer pairsin a multiplex PCR reaction, as well as a separate PCR reaction forprimer pair IKB5A/B. To ensure equal quantities of all amplicons in themultiplex reaction, primer concentrations were varied. Primer pairsIK25A/B, IK114A/B, IK123A/B, and IK127A/B were used at a concentrationof 200 pmoles per primer; primer pairs IK8A/B, IK118A/B, and IKB3A/Bwere used at 100 pmoles per primer. In the separate PCR reaction, primerpair IKB5A/B was used at a concentration of 200 pmoles per primer. Theseamplicons were purified, labeled with the ECL kit and pooled; theseconstituted the “probe-amplicons”. Each membrane strip containing thetarget-amplicons was hybridized at 42° C. with the pool of purifiedprobe-amplicons generated from a single isolate and autoradiographsprepared by exposure of processed blots to the Kodak Scientific ImagingX-OMAT AR film (Eastman Kodak Company), to detect the presence orabsence of hybridizing amplicons in the isolates being analyzed.

[0159] Software.

[0160] PFGE gels were analyzed using Molecular Analyst FingerprintingPlus software (Bio-Rad). Dendrograms were constructed using theunweighted pair-group method with arithmetic mean (UPGMA).

Example 3

[0161] Insertions Deletions, and SNPs at AvrII Sites Enhanced the PATSStrain Typing System for E. coli O157:H7

[0162] We designed primer pairs to amplify DNA flanking 33 sites in theO157 genome for the rare cutting restriction enzyme, AvrII. Of these 33sites, 7 sites were identified that were polymorphic between O157isolates. In the case of the AvrII sites, polymorphisms were due toeither insertions, deletions, or single nucleotide polymorphisms (SNPs).The SNPs occurred either within the AvrII site itself, resulting in lossof the site, or in sequences near the site, resulting in the creation ofan additional AvrII site.

[0163] Of the 7 polymorphic AvrII sites, 5 were in O-islands and 2 werein the backbone (sequences shared with E. coli K12). Adding primer pairsspecific for DNA flanking these 7 polymorphic AvrII sites to the primerpairs specific for the 8 polymorphic XbaI sites and four virulence genes(stx₁, stx₂, eae, hlyA), made the PATS typing system highlydiscriminatory for distinguishing strains of O157.

[0164] The primer pairs depicted in Table 6 produced polymorphic resultsacross the isolate set, amplifying products with an AvrII site in someisolates but failing to amplify any product in others. TABLE 6 DistanceAmplicon No. Seq ID Primer Length Sequence (5′→3′) From AvrII Size Tm(° C.) 1 29 IKNR3 A 24 GCACCATTCATGATATTCGTTAAC 254 bp 380 bp 66 30IKNR3 B 24 TTGCAATGTTCATTAATATACGTC 126 bp 62 2 31 IKNR7 A 24TATACTCATTGATAAAATACTAAC 268 bp 406 bp 58 32 IKNR7 B 24AGCACAGAAGAGTAATTATATGTC 138 bp 64 3 33 IKNR10 A 24ATCAGGATGCCGTFATACTCATTG 282 bp 419 bp 68 34 IKNR10 B 24GCACAGAAGAGTAATTATATGTCC 137 bp 66 4 35 IKNR12 A 24AAGTTTTGATATTGTACTGGATGC 304 bp 443 bp 64 36 IKNR12 B 24CATTAAAGATAGATGATAAATCAC 139 bp 60 5 37 IKNR16 A 24TGCTCAACATAGAAACCCACATAG 144 bp 444 bp 68 38 IKNR16 B 24TCGAATCAGTGTTATTTACCAGTG 300 bp 66 6 39 IKNR27 A 24GTTATTCTGGTACATGAACATCAT 336 bp 524 bp 64 40 IKNR27 B 24TAGATAATTCCACACAGCCCACTA 188 bp 68 7 41 IKNR33 A 24GTAGTCGAAATCATGGTGCAGAAT 217 bp 383 bp 68 42 IKNR33 B 24CTTCTCTGCTGTTTGGTGTCTTAT 166 bp 68

[0165] The DNA sequences amplified by the AvrII primer pairs wereanalyzed using the Genbank database (BLAST search program, NCBI) and theE. coli O157:H7 strain 933 genome sequence database (University ofWisconsin). Of the 32 AvrII-containing genome sequences analyzed, 22were homologous to E. coli strain K-12 genome sequences (referred to asbackbone sequences (Perna et al., Nature 409, 463-466 (2001)), while 10were in regions of the O157:H7 chromosome not shared with K-12, referredto as O-islands (SEQ ID NO.: 1) (Perna et al., Nature 409, 463-466(2001)). The majority of the polymorphic regions were localized to theO-islands (5/7), compared to a few in the conserved regions (5/25)indicating again that genetic differences between E. coli O157:H7strains occur in O-islands. The location of the regions amplified byeach primer pair is shown in Table 7. TABLE 7 E. coli O157 genomicregions amplified by the 32 PATS primer pairs designed flanking AvrIIrestriction enzyme sites. Regions conserved Regions polymorphic acrossall strains between strains Location in the Location in the O157 Primerpair O157 genome Primer pair genome IKNR1A/B Backbone^(a) IKNR3A/BO-island IKNR2A/B Backbone IKNR7A/B O-island IKNR4A/B Backbone IKNR10A/BO-island IKNR5A/B Backbone IKNR12A/B O-island IKNR6A/B O-island^(b)IKNR16A/B Backbone IKNR8A/B O-island IKNR27A/B Backbone IKNR9A/BO-island IKNR33A/B O-island IKNR11A/B Backbone IKNR13A/B O-islandIKNR14A/B Backbone IKNR15A/B Backbone IKNR17A/B Backbone IKNR18A/BBackbone IKNR19A/B Backbone IKNR20A/B Backbone IKNR21A/B BackboneIKNR22A/B O-island IKNR23A/B Backbone IKNR24A-1/B-1 Backbone IKNR25A/BBackbone IKNR26A-1/B-1 Backbone IKNR28A/B Backbone IKNR30A/B BackboneIKNR31A/B Backbone IKNR32A/B Backbone

[0166] PATS profiles of O157 strain isolates were also identified usingprimers that flanked AvrII restriction sites and virulence gene primerpairs. Table 8 shows the result of this analysis. TABLE 8 PATS profilesof O157 isolates based on AvrII restriction sites and virulence genes.PCR amplification and AvrII restriction digestion patterns of ampliconsobtained using 31 PATS - 4 virulence gene primer pairs^(b) PATS type^(a)IKNR1 IKNR2 IKNR3 IKNR4 IKNR5 IKNR6 IKNR7 IKNR8 IKNR9 IKNR10 IKNR11IKNR12 IKNR13 Control 2 2 2 2 2 2 2 2 2 2 2 2 2 Control 2 2 2 2 2 2 2 22 2 2 2 2 A(3) 2 2 2 2 2 2 2 2 2 2 2 2 2 B(1) 2 2 2 2 2 2 2 2 2 2 2 0 2C(2) 2 2 2 2 2 2 2 2 2 2 2 0 2 D(2) 2 2 2 2 2 2 1 2 2 1 2 2 2 E(4) 2 2 22 2 2 1 2 2 1 2 2 2 F(4) 2 2 2 2 2 2 2 2 2 2 2 2 2 G(2) 2 2 1 2 2 2 2 22 2 2 2 2 H(2) 2 2 2 2 2 2 2 2 2 2 2 2 2 I(2) 2 2 1 2 2 2 2 2 2 2 2 2 2J(1) 2 2 2 2 2 2 2 2 2 2 2 2 2 K(21) 2 2 2 2 2 2 2 2 2 2 2 2 2 PCRamplification and AvrII restriction digestion patterns of ampliconsobtained using 31 PATS - 4 virulence gene primer pairs^(b) PATS type^(a)IKNR14 IKNR15 IKNR16 IKNR17 IKNR19 IKNR20 IKNR21 IKNR22 IKNR23 IKNR24IKNR25 Control 2 2 2 2 2 2 2 2 2 2 2 Control 2 2 2 2 2 2 2 2 2 2 2 A(3)2 2 2 2 2 2 2 2 2 2 2 B(1) 2 2 2 2 2 2 2 2 2 2 2 C(2) 2 2 2 2 2 2 2 2 22 2 D(2) 2 2 1 2 2 2 2 2 2 2 2 E(4) 2 2 1 2 2 2 2 2 2 2 2 F(4) 2 2 2 2 22 2 2 2 2 2 G(2) 2 2 1 2 2 2 2 2 2 2 2 H(2) 2 2 2 2 2 2 2 2 2 2 2 I(2) 22 1 2 2 2 2 2 2 2 2 J(1) 2 2 2 2 2 2 2 2 2 2 2 K(21) 2 2 2 2 2 2 2 2 2 22 PCR amplification and AvrII restriction digestion patterns ofamplicons obtained using 31 PATS - 4 virulence gene primer pairs^(b)PATS type^(a) IKNR26 IKNR27 IKNR28 IKNR30 IKNR31 IKNR32 IKNR33 stx₁ stx₂eae hlyA Isolates^(c) Control 2 2 2 2 2 2 2 1 1 1 1 E. coli O157:H7strain EDL933 Control 2 3 2 2 2 2 0 0 1 1 1 E. coli O157:H7 strain 86-24A(3) 2 3 2 2 2 2 0 0 1 1 1 G5290, G5311, G5312 B(1) 2 3 2 2 2 2 0 0 1 11 G5289 C(2) 2 2 2 2 2 2 2 1 1 1 1 G5316, G5320 D(2) 2 2 2 2 2 2 2 1 1 11 G5324, G5325 E(4) 2 2 2 2 2 2 2 0 1 1 1 G5283, G5284, G5307, G5308F(4) 2 3 2 2 2 2 2 1 1 1 1 G5291, G5292, G5303, G5304 G(2) 2 2 2 2 2 2 20 1 1 1 G5295, G5296 H(2) 2 3 2 2 2 2 0 1 1 1 1 G5305, G5306 I(2) 2 2 22 2 2 2 1 1 1 1 G5317, G5318 J(1) 2 2 2 2 2 2 2 1 1 1 0 G5323 K(21) 2 22 2 2 2 2 1 1 1 1 G5285, G5286, G5287, G5288, G5293, G5294, G5297,G5298, G5299, G5300, G5301, G5302, G5309, G5310, G5313, G5314, G5315,G5321, G5322, G5326, G5327

[0167] PATS amplicon analysis using AvrII and the virulence gene primerpairs identified eleven different PATS profiles (Table 8) for O157isolates, compared to the fourteen PATS profiles (Table 5) obtained forthe same set of isolates using XbaI and the virulence gene primer pairs.However, PATS amplicon analysis using XbaI, AvrII, and the virulencegene primer pairs was able to discriminate 20 different PATS profilesfor the same O157 isolates. The results of this analysis are shown inTable 9. TABLE 9 PATS profiles of O157 isolates based on polymorphicXbaI and AvrII restriction sites, and virulence genes PCR amplificationand restriction digestion patterns of amplicons obtained using 15 PATS -4 virulence gene primer pairs^(b) Polymorphic Polymorphic XbaI sitesAvrII sites PATS type^(a) IK8 IK19 IK25 IK114 IK118 IK123 IKB3 IKB5IKNR3 IKNR7 IKNR10 IKNR12 Control 0 2 0 2 2 2 2 2 2 2 2 2 Control 2 2 20 2 2 0 2 2 2 2 2 A(3) 2 2 2 0 2 2 0 2 2 2 2 2 B(1) 0 2 0 2 2 2 2 2 2 22 2 C(1) 0 2 0 2 2 2 2 2 2 2 2 0 D(1) 2 2 2 0 2 2 0 2 2 2 2 0 E(1) 0 2 02 2 0 2 2 2 2 2 2 F(1) 2 2 0 2 2 2 0 0 1 2 2 2 G(2) 2 2 0 2 2 2 0 0 2 11 2 H(4) 2 2 0 2 2 2 0 0 2 1 1 2 I(2) 2 2 0 2 0 2 0 0 1 2 2 2 J(2) 2 0 02 2 2 2 2 2 2 2 2 K(1) 0 2 0 2 2 0 0 2 2 2 2 2 L(1) 2 2 0 2 2 0 0 2 2 22 2 M(2) 2 2 2 0 2 0 0 2 2 2 2 2 N(2) 2 0 0 2 2 2 0 2 2 2 2 2 O(1) 2 2 02 2 2 2 0 1 2 2 2 P(1) 2 2 0 2 2 2 0 2 2 2 2 0 Q(2) 2 2 0 2 2 2 0 2 2 22 2 R(6) 2 2 0 2 2 2 0 2 2 2 2 2 S(10) 2 2 0 2 2 2 2 2 2 2 2 2 PCRamplification and restriction digestion patterns of amplicons obtainedusing 15 PATS - 4 virulence gene primer pairs^(b) Polymorphic VirulenceAvrII sites genes PATS type^(a) IKNR16 IKNR27 IKNR33 stx₁ stx₂ eae hlyAIsolates^(c) Control 2 2 2 1 1 1 1 E. coli O157:H7 strain EDL933 Control2 3 0 0 1 1 1 E. coli O157:H7 strain 86-24 A(3) 2 3 0 0 1 1 1 G5290,G5311, G5312 B(1) 2 2 2 1 1 1 1 G5327 C(1) 2 2 2 1 1 1 1 G5320 D(1) 2 30 0 1 1 1 G5289 E(1) 2 2 2 1 1 1 0 G5323 F(1) 1 2 2 1 1 1 1 G5317 G(2) 12 2 1 1 1 1 G5324, G5325 H(4) 1 2 2 0 1 1 1 G5283, G5284, G5307, G5308I(2) 1 2 2 0 1 1 1 G5295, G5296 J(2) 2 2 2 1 1 1 1 G5288, G5299 K(1) 2 32 1 1 1 1 G5303 L(1) 2 3 2 1 1 1 1 G5304 M(2) 2 3 0 1 1 1 1 G5305, G5306N(2) 2 2 2 1 1 1 1 G5313, G5314 O(1) 1 2 2 1 1 1 1 G5318 P(1) 2 2 2 1 11 1 G5316 Q(2) 2 3 2 1 1 1 1 G5291, G5292 R(6) 2 2 2 1 1 1 1 G5297,G5298, G5301, G5302, G5309, G5310 S(10) 2 2 2 1 1 1 1 G5285, G5286,G5287, G5293, G5294, G5300, G5315, G5321, G5322, G5326

[0168] The results of these analyses are also represented asdendrograms. FIGS. 9A, 9B, and 9C show dendrograms based on PATSprofiles from XbaI primers, AvrII primers, and a combination of XbaI,AvrII, and virulence gene primers, respectively.

[0169] Detailed Materials and Methods

[0170] Described below are detailed materials and methods relating tothe above-described experiments. In the case of the AvrII sites,polymorphisms were due to insertions, deletions, or single nucleotidepolymorphisms (SNPs). The SNPs occurred either within the AvrI siteitself, resulting in loss of the site, or in sequences near the site,resulting in the creation of an additional AvrII site.

[0171] Design of Primer Pairs Amplifying 0157 AvrII Site

[0172] The sequenced EDL 933 genome (GenBank accession number AE005174;Perna et al) was used as the prototype to determine the total number ofAvrII restriction sites, and the DNA sequence of the regions flankingthese sites in an O157 genome. This sequence was used to design 32primer pairs that would yield distinct amplicons containing a singleAvrII site from O157 strain EDL 933. The primers were assigned a prefixIKNR.

[0173] PCR Conditions for AvrII Primer Pairs

[0174] PCR was carried out using conditions described previously (Kudvaet al., J Clin Microbiol. 40:1152-9, 2002; Kudva et al., J Bacteriol.184:1873-9, 2002). Briefly, colony lysates were prepared by boilingcolonies suspended in sterile distilled water, followed bycentrifugation at 4° C. Each O157 strain template was tested with eachindividual primer pair. PCR was carried out on the GeneAmp PCR system2400 thermal cycler (PE Biosystems, Foster City, Calif.), using 10 μl ofcolony lysate, 200 pmoles of each primer, 800 μM dNTPs, 1× diluted ExTaq™ enzyme buffer and 2.5 units of TaKaRa Ex Taq™ DNA polymerase. Thehot start PCR technique (Dieffenbach et al. PCR Methods Appl. December1993;3(3):S30-71) was employed in combination with a touchdown PCRprofile (Don et al Nucleic Acids Res. Jul. 25, 1991;19(14):4008). Tocreate this profile, an amplification segment of 20 cycles was set wherethe annealing temperature started at 73° C., to touchdown at 53° C. atthe end of those cycles. Subsequently, another amplification segment of10 cycles was set, using the last annealing temperature of 53° C. Eachreaction was done in triplicate.

[0175] Evaluation of AvrII Amplicons

[0176] PCR reactions were initially screened for the presence or absenceof amplicons. Amplicons, when present, were purified using the QiaquickPCR purification kit and digested with AvrII to confirm the presence ofan AvrII site within the amplicon. Undigested and digested DNA fragmentswere resolved on a 4% agarose gel prepared with a combination of 3%NUSIEVE GTG agarose (FMC BioProducts, Rockland, Me.) and 1% agarose(Shelton Scientific Inc., Shelton, Conn.) and stained with ethidiumbromide.

[0177] DNA Extraction, Sequencing and Probe Labeling

[0178] Genomic DNA was prepared using the INVITROGEN EASY-DNA ISOLATIONKIT (Invitrogen Corporation, Carlsbad, Calif.) as per the manufacturer'sinstructions. DNA sequencing was done at the DNA Sequencing CoreFacility, Department of Molecular Biology, Massachusetts GeneralHospital. All DNA probes were detectably labeled using the ECL DIRECTNUCLEIC ACID LABELING AND DETECTION SYSTEM (Amersham Pharmacia Biotech,Inc., Piscataway, N.J.).

[0179] Southern Blot

[0180] DNA was fractionated by agarose gel electrophoresis, transferredto HYBOND-N+ nitrocellulose membranes (Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.), crosslinked to the membrane using ultraviolet lightin a STRATALINKER (Stratagene), and hybridized with the appropriateprobe, which was detectably labeled using the ECL DIRECT NUCLEIC ACIDLABELING AND DETECTION SYSTEM (Amersham Pharmacia). Hybridization at 42°C. and post-hybridization washing of blots was done according to themanufacturer's instructions. Autoradiographs were prepared by exposureof processed blots to Kodak Scientific Imaging X-OMAT AR film (EastmanKodak Company, Rochester, N.Y.).

[0181] Data Analysis for AvrII Amplicons.

[0182] Statistical analysis was performed using Epilnfo6 (available fromthe Center for Disease Control) software. The significance ofdifferences in proportions was calculated with the Fisher's exact test.DNA G+C content was determined using the Wisconsin Package Version 10.0,Genetics Computer Group (GCG), Madison, Wis.

Other Embodiments

[0183] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 42 <210> SEQ ID NO 1<211> LENGTH: 61662 <212> TYPE: DNA <213> ORGANISM: Escherichia coli<400> SEQUENCE: 1 catgtctaag gatatggttt atcattttga aaaatataat tttatttcatcctcctggtc 60 actttggggc acgtctgggg cacgggcatt aaggacatta ttcaacatggcaacttgagt 120 cacgctgcac tcaggcatcc atgcaccata aacattgtag accatgctggcgctggagtg 180 ccccatctgt gatgcaataa atgtcgggtt tgctccggaa gataaagcccagcacgcata 240 ggtatggcgt gactgatacg ctttacggga tcggataccc gctctttttattgctgaatc 300 ccatgtcgct ccgatggagc ttaccgcgta gttaataccc gccttgtgattcttgcgaac 360 gatttgcgga cagaaaacaa aagtgcactc gtgcaaaatt gttcttccgtactcgcgtaa 420 ttgaacagtg atctgatgct gcctgctaag acgagtaagc atcgcctggtttttaagtgc 480 ttcaattgct ggtgccagaa gatgtataac ccggttagtg cctgcgtcggtctttggtag 540 cgtaaaatca cctatttttg taaaatttcg tcgcactgtt atcgtgccagctttcaggtc 600 gatatcctcc catgcaagtg cggcaatttc accgtgtcgc atccctgtaaaaacagccac 660 tgtccagagg tttttggtct gttgatgatg gcaggcatca atgagacggctaaactcatc 720 tctggtcagt ggatccggca ctggttttga tttcctcagc ggtgttattgaattaaacgg 780 gtttttctcc agatacccgt tttcggcggc aaagctgaac attccggctgttgttgtcat 840 gtaatagttc actgtgggta cggttcttcc ttttcgggac gtgctggttttcctgcttcc 900 cttttccccg gtcagtaaat ctttcctgat aaacagcaga tcttcttttgtaatcgatga 960 cgccagcctg ccaggaccaa gcctcggtag catatttttc atcactgattgataacgatt 1020 taaggcgtta ctaccgattt ccattgcttt cagagtaagc catttctgtgccagttcacc 1080 gacggtgata tcttttttta ccaggccaaa tagcttcagg ttaggtgagtcagggaatcg 1140 atcggcataa tcaaacgttc ctgttctgat tgcaaagcag accgaagcccttaactcacc 1200 agcgatcttt ctgtttttcg gtgtatcggg cacgcgaaga ttttcgcgcactcgtttacc 1260 cctgtactta aacgttattc ggagttttcc tccgtgattt tcaacgccggctggataggc 1320 tgaattcgcc attgttcctc ctgcgtccaa gagcacgctc aggttatactcttatggaca 1380 agaattaaac atcctgagat ggtaagggtt ggtttttgat ccaagtgttgatggtaggaa 1440 gatgatagag acattcgctg tttttctttg gcgttccgtc gggggcgatttgtttatatt 1500 ctctgccgtt catccacgca ctgtccctgg cgcgtaatat tgttccttttctcaggcctg 1560 ttacagccat taaaagctcc agtgttaccc attcattcgc atttatctgtacaacaggtg 1620 tgatgactgg ctgaatgcca tggttatgtt ggtttactaa ctcgcgcataagttgcctct 1680 gcatgtgtaa gaaaaaaccg ccatcaggcg gcttggtgtt ctttcagttcttcaattcga 1740 atattggtta cgtcttattc gatgcgcact cctggtattt cgccttttgatattgctaag 1800 tcataaattt gcgcggcact atacccatct cgcatccatg aatctaaggcgcgaacagcc 1860 tcgctacgct ttttatcttc tctctcattt ttgatatcaa cgaggacatcaacgcaatta 1920 aggcaaatgt ggattttgtc tttacattcg atcatggcgg ctttgccatgatttccgcca 1980 cacagtgagc ataaatcttc agggtctggc tggtatttct gtaacgttagagggttgaat 2040 gttgaacagg ccataatcat ctccataaaa caaaactcgc cgtagcgagttcagataaaa 2100 gaaatccccg cgagtgcgag gattgttatt cattgccgat attcacctttatcgcgaaca 2160 cctttaccgg tttatcaccg aagtgcggat gtgtgattgt cttgatttcatatccgtcat 2220 acggaacatc aattcttcgg ctggaatcgt cgcgcttcgg atatccctttgtgataatca 2280 ggcggtcata ctcgcggaac ataattcgct tattccagta gtcattaaacaggcgatact 2340 cttccgtttt ctctccgcga atcatggcat cgaagtattc acctttaacggcaagttgca 2400 ggttagccac ggttaacctc ctgcggcggt tctggtagcg gcatccagtgagttgcttgc 2460 tcaataccat tacccggctt aatcgttgca tctccgcgcc gaaaggtgcttccggtatag 2520 cgtgcggagc atattagcgg ttcaaccaga gagctatcga aattcaccgaaataagcacg 2580 ttctggccct tttcaggcat tcgatcacta cagcttatcc aactatccggagttaccgga 2640 gagttaccag cctcataagc ggatttcatc cagtgcgtaa gcgtttcgatgcttacacat 2700 ccgcaatcaa cgtctatttt ttctttttgt tctgacaacc attcctggaatgacagcttg 2760 gcagtctggc ttgctggatc aattcgtggc aggccgatat atagtggtacatttcccggc 2820 tccatcgaat tgtcgggaca aataaacgtg ttacaaccat atttaacgagctcaattccc 2880 actgtgtcga tagtggcgaa tggttcagtg gtcaatgcag tcaacgcaatttcataagca 2940 cggcgctcaa tattatctcg cacgtccagg ctgccgattc gctctttgatttctttaatc 3000 atttctttgt cggtgaacgt tgtcatgtgt tagtccttat ccacttcaacgccatctttc 3060 agcgtgatgc cgtgccaatc atcagcccaa ctggttaacc caggcgcatcaatgctaggc 3120 atatagacgc ttgcagtgtg gtagccctta tcgttatcaa tgctggcaacgtgctcgccg 3180 ttgtatgcgc tcagcgtgtc taggacacta taaaactttc ctccggctgccctgaaatcc 3240 tttacagcct tcacaagacg attccacgct ttttcctgtt ctggcgtcaggtcgattaat 3300 tcctgcaaag ttgccatttc agttttcctt atatgggtta attttattgtgcagtgtgtt 3360 gaacgacgcc cataccacgt cgttatacaa ctcaataact agctcaattattttcccgat 3420 tgcccagaca aaaattagcg gggatatcgg tgtcatcaac acgataaacagaatgagaaa 3480 caaaaattct gtcgctctac tttttcgcgg atattctttt ctgaataatgtaggcacatc 3540 actctccttt gttgctcctc aaaattttat gccctggcgc aaaagcacgcgttttgtctt 3600 tgcttattcg ccagccatcc ttgcgcgcct cttttgcaca gccagcccatgacgtaccga 3660 tatactcacc gaagtctggc gacttatatt tgccatctgt acactggaggcaatcacaat 3720 agagatgcat ggtgtaactt gcagcaatag ccatatcact ctcctttagtgcgcaagtgg 3780 tttttccagc ggttttgctc cgcgctgggc tttttgcaaa aaccacaatccatcatcccg 3840 taatatttca tcaaccccat ccgtcggttg ctgagtctca cccactgccagacgccagga 3900 gcgtttctac gaactaacag aatctttgct ttacggtttt tcatcgttttgctctcctgc 3960 atctctttgc tgctcgtcgt gccgctgcaa taccggtatg gcggcgctttggtgtcggga 4020 tgatgttctt tgcaattagc gcagaagccc aaaaacgagt cggatacggtaacaagccga 4080 tacatgccac acgcattact cacctctttt gatgcgaatg ccagcggcgcgctcggcttc 4140 actttgttcc caaaaccact tgtgaagcgc cataagcttt tcgtcaatcggtgcatattt 4200 gcgattaaag taggcctgag catctttctc agattcgtcc ggtaattcgccagggccaaa 4260 cagtgtgtta taaatccatg ctagtccgct cttagcgtcg ccagttgcctgccattcgat 4320 aatggcagcc tgcatgacca gaatgttttt cccgattaat aggtccagttctttgtaccg 4380 gttgcggatg tatgcattct cgctttgtaa ttttgcgttg cgcttttctgaggcttcaag 4440 taacgcctgc ttatcgcgta gagcttcttc cagttcagca acatggcattcactatcaat 4500 aaggttgttc tctgctgctt caagctcaac acgcagcttc ccaaccgtaagcgcaatctc 4560 ctcgttctcc tggtcgcggc gtttgatgta ttgctggttt ctttcctgttcatccagcag 4620 tgccagcacg gtagccgggt tagcctctgc tatgaattca gcgtttgcataagcctgatc 4680 atctgattca atcaggcagt taacatgaca ttccgcaatc acgccaccgggttctccttt 4740 ccatttttgg caaacaaaaa ctcctgttaa attgccgtgc tggttaacagatgtatgccc 4800 tacgatgtag cttcctttag ttgctttctc tgccttttca cgcagtgcctgataattaat 4860 ttcgctcact tcgaacctct ctgtttactg ataagctcca gatcctcctggcaacttgca 4920 caagtccgac aaccctgaac ggccaggcgt cttcgctcat ctatgggatcgccacactca 4980 caacaatgag tggcagatat agcctggtgg ttcaggcggc gcatttttattgctgtgttg 5040 cgctgtaatt cttcaatttc tgatgctgaa tcaatgaggt ctgccatctttcattaatcc 5100 ctgaattgtt ggttaatacg cttgagggtg aatgcgaata ataaaaaaggagcctgtagc 5160 tccctgatga ttttgctttt catgttcacc gttccttaaa gacgccgtttaacatgccga 5220 tcgccagact taaatgagtc ggtgtgaatc ccattagcgt taccgtttcgcggtgcttct 5280 tcagtacgct acggcaaatg tcatcgacgt ttttatccgg aaactgctgtctggcttttt 5340 tgatttcaga attagcctga cgggcaatgc tgcgaagggc gttttcctgctgaggtgtca 5400 ttgaacaagt cccatgtcgg caagcataag cacacagaat atgaagcctgctgccagaaa 5460 aatgcattct gttgttgtca tgccgggtct ctctcgtttg cttctgctttcgccgccatc 5520 atttccagct tttgtgaaag ggatgtggct aacgtatgaa attcttcgtctgtttctact 5580 ggtattggca caaacctgac tccaatttga gcgaggctat gtgccatcccgatactcgtt 5640 cttaattcaa caggagatgc tttgtgcata cagcccctcg tttattatttatctcttcag 5700 ccagccgctg tgctttcagt ggatttcgga taacagaaag gccgggaaatacccagcctc 5760 gctttgtaac ggagtagacg aaagtgatcg tgcctacccg gatattatcgtgaggatgct 5820 tcattaccat tgctccccat atacaaaacc aatttcagcc agtgcctcgtccattttttc 5880 gatgaactcc ggcaccatct cgtcaaaact cgccatgtac ttttcattccgctcaatcac 5940 gacataatgc aggccttcac gcttcatgcg cgggtcatag ttggcaaagtaccaggcatc 6000 ttttcgtgtc acccacatgc tgtactgcac ctgggccatg taagccgattttatggcctc 6060 gaaaccaccg agccggaact tcatgaaatc ccgggaggta aacgggcatttcagctcaag 6120 gccgttgccg tcactgcata aaccatcggg agagcaggcg gtgcgcatactttcgtcgcg 6180 atagatgatc ggggattcag taacattcac gccggaagtg aactcaaagagggttctggc 6240 gtcgttctcg tactgttttc cccaggccag agccttagcg ttaacttccggagccacacc 6300 ggtgcaaacc tcagccagca gggtgtggaa gtaggacatt ttcatgtcaggccacttctt 6360 tcctgatcgg ggttttgcta ttacgttgtg aatttctgaa gctgtgatgacgccgagccg 6420 taatttgtgc cacgcatcat ctccctgttc gacagctctc acgtcgatcccggtacgctg 6480 caggataatg tccggtgtca tgctgccacc ttctgttcag tggctttttgtttcaggaat 6540 ccaagagctt ttactgcttc ggcctgtgtc agttctgacg atgcacgaatgtcgcggcga 6600 aatatctggg aacagagcgg caataagtcg tcatcccatg ttttatccagggcgatcagc 6660 agagtgttaa tttcctgcat ggtttcatcg ttaaccggag tgatgtcgcgttccggctga 6720 cgttctgcag tgtatgcggt attttcgaca atgcgctcgg cttcatccttgtcatagata 6780 ccagcaaatc cgaaggcgag acgggcacac tgaatcatgg ctttatgacgtaacatccgt 6840 ttgggatgcg actgccacgg ccccgtgatt tctctgcctt cgcgggttttgaatggttcg 6900 cggcggcatt catccatcca ctcggtaacg cagatcggat gattacggtccttgcggtaa 6960 atccggcatg tgcaggattc attgtcctgc tcaaagtcca tgccatcaaactgctggttt 7020 tcgttgatga tgcgggacca gccatcaacg cccaccaccg gaacgatgccgttctgctta 7080 tcagggaagg cgtaaatttc tttcgtccac ggattaaggc cgtactggttggcgacgatc 7140 agcaatgcga tgaactgcgc atcgctggca tcacctttaa atgccgtctggcgaagagtg 7200 gtgatcagtt cctgtgggtc gacagaatcc atgccgacac gttcagccagcttcccagcc 7260 agcgttgcga gtgctgtact catccgtttt atacctctga atcaatatcaacctggtggt 7320 gagcaatggt ttcaaccatg taccggatgt gttctgccat gcgttcctgaaactcaacat 7380 cgtcatcaaa cgcacgggta atggcttttt tgctggcccc gtggcgttgcaaatgatcga 7440 tgcatagcga ttcaaacagg tgctggggca ggcctttttc catgtcgtctgccagttctg 7500 cctctttctc ttcacgggcg atctgctggt agtgacgcgc ccagctctgagcctcaagac 7560 gatcctgaat gtaataagcg ttcatggctg aactcctgaa aatggctgtgaaaatatcgc 7620 ccgcgaaatg ccaggctgat taggaaaaca ggaaatgggg ttagtgaatgcttttgcttg 7680 atctcagttt cagcattaat atccattttt tataagcgtc gacagcttcacgaaacatct 7740 tttcatcgcc aataaaagtg gcgatagtga atttagtctg gatagccataagtgtttgat 7800 ccattctttg ggactcctgg ctgattaggt atgtcgataa ggcgtttccatccgtcacgt 7860 aatttacggg tgattcgttc aagtaaagat tcggaagggc agccagcaacaggccaccct 7920 gcaatggcat attgcatggt gtgctcctta tttatacata acgaaaaacgcctcgagtga 7980 agcgttattg gtatgcggta aagccgcact caggcggcct tgatagtcatatcatctgaa 8040 tcaaatattc ctgatgtatc gatatcggta attcttattc cttcgctaccatccattgaa 8100 ggccatcctt cctgaccatt tccatcattc cagtcgaact cacacacaacaccatatgca 8160 tttaagtcgc ttgaaattgc tataagcaga gcatgttgcg ccagcatgattaatacagca 8220 tttaatacag agccgtgttt attgagtcgg tattcagagt ctgaccagaaattattaatc 8280 tggtgaagtt tttcctctgt cattacgtca tggtcgattt caatttctattgatgctttc 8340 cagtcgtaat caatgatgta ttttttgatg tttgacatct gttcatatcctcacagataa 8400 aaaatcgccc tcacattgga gggcaaagaa gatttccaat aatcagaacaagtcggctcc 8460 tgtttagtta cgagcgacat tgctccgtgt attcactcgt tggaatgaatacacagtgct 8520 tattcgtact aataaaatac ccaattttct gtttcttggt tgtgtccaaagttatattca 8580 atatctggtg ttgatgtatc aatattcttc ataccatcaa caagagttgatacaacagcc 8640 aaatcttgtt tgattctcat taaatggtat ttcttccggc gcaataaactttcaatggca 8700 agtttcttcg ttgggaatgc aaaagatctt tctgcatttt ttgctactttcttaattgca 8760 tatctatttc tcctttgttt ccattcctgt aaccactgat ttggtgctggtttaaaatta 8820 acaatccaat gcgcaggaac caaccatgca taatgctctg tctgatgaaaagctatatat 8880 tgaagtgcga atatttttat cccatcttct tcaactgtcg cctggaatctccagaaaaca 8940 ggcattccat catgttcagt ttctgattca ggaaaaggta cgctccatgattttgtcata 9000 tctcacctca aataagtggt ttgctgccta atttcatttt ctggcgaccaacacaagtca 9060 cctcgccgtc agttgttttg atttccggta gcctgccgcg taaatggctacgtttggaag 9120 acatacacca gtttctggtt gcttatgtcc aaactcattc gcgtacacaatggccgctcg 9180 ctccagattg cgtctgtatt ctttctgttg ccagatcacg tcctgtgccatgaacttaat 9240 tggcttagcg tcttctatgc gctcaggcgt ttcgtgagta cctttagcctgaatctgcgc 9300 tctgcttaga gtagggcggt gtaatacttc tgaacttatt gcttcttcgcgggccagtac 9360 gccgttagct aatgcctttg cctttaaacg ctcacgacga cgagaacgtgaattgccttt 9420 gaactgagtt ctgcgtgtca tatagacctc ctgatgaact ttggtggtgtggtaggtggg 9480 agacccattt cgacctgttt cggcctactt caattcggca atagtcccgcaggcctcgcc 9540 gctttacgtg cgacatattc ccgtccatga acccttcacc acaccccaaagttcactttg 9600 gttattgcgc tttgtcagcg ccgtagattc atattcgaat cgttgtatattcaccgccct 9660 ggtgagtaat gcgtcctgct gacgacgata ataatgaacc aatagttcgacattatcaag 9720 aactattggt acgaattttg gtgatttatt aactctacga agtatgattctgatatataa 9780 ggaaatttat ttttgaaaat gtggctgatg aaggttatgc ggcagggatcataactgcat 9840 ggtttagcga gttacatcaa taaatacaat tggttatgtt ttttaggtgggcgaacgtga 9900 ggcaaagaaa acccggcgct gaggccgggc tagattttaa agtatttatcttttagagat 9960 gtagatgtaa aacttttcgc ctttgaaaat tttttgtcat cagaagggcttatgaactca 10020 tcttttttgt agggaaccgc taatgctgca tcacgtctgc gaggcagcttgcttacttcc 10080 tcgcgctttt tcatgatcag ttatccttta ataacctata cagttttgtaggggtacatc 10140 ctgaggatat tgttaagttc gtagcacgcc ttttccgccc atcatcgtataaacgaaaac 10200 cagtagtaga cgaattttct gcgtcaaaaa ctatagacag tatagcgtccccagactttt 10260 tttgccattc gcatgtgccg ttagttggtt tcgtcatctg tagacgccagtcaagaacgc 10320 catcacttat agctgagaga tcgtttagta catctagtac ggattgatatctttcatttg 10380 gatctacatg aatgcatttg ttcactattg ttattaattt tttatgtatatggggaggat 10440 actcttttaa tggatagcag ccattaatta tcgactctct gagttgttcaatcgtgctaa 10500 atgcagatct ttctctttca aaattatcat gtccaacaca cattctatatatggttaatc 10560 ctgcctgata tatgtcatat gtgaaattat aatcatttgt tgataaagaaaaatattccg 10620 gtggcacatg aaaatgatat ccaaactcag gcgcagctct cgattcctcattgactaact 10680 gagataatcc aaagtcagat agcatggcct catttctgtt tgatatcataatgttattag 10740 gttttatatc aaaatgcata agaccttttg agtgtatatg ataaagtccacttaaaaatt 10800 gaatggaata ccgtattatc tccctgcttg taagattatt ttttttcattaattggttta 10860 gcgaaccatt atgataaaat ggcatggcta tatagatatt gctctcacattgagcagcat 10920 actgaacttg cacaatattt ggatgtgcat gtttatagag aagccttgcttcattaaagt 10980 agtcgtcgtg gttagtgttt tctttttttt ctatttcttt aatcaccaagtcatgagcta 11040 ggtgtctgtc atgagccaga tatacttttg aaaaacaacc ctgttcttctagatcactaa 11100 tccattcgaa ttctacatca gctcttttgt atggagttag catccccttacctccgcaga 11160 tagtgcagcc aaaacagctt catttgtttc agttgtaaaa ccagaattatcgattccatt 11220 tatattgcgg tgtgacttca atatttcttt atactcgatc tctgttaggttcaatgatga 11280 tttcatgcca gattttctaa tagtgtaata ttttcttaca tcactgcttgaaaatgcttc 11340 ttgaataaca gcttctatat aaaggcggtc aatgctaaga ttatcagagtttgattcagt 11400 aacgcgtatg gcagctaatt caacattata taaattaaga atgtcgaggatgttatttct 11460 cacatacttt aatttttctg gtgtgtctaa ggtcgaaggt attttaataacatcaacaca 11520 tttgagtgca gactcattag tgcaatatac aacaaaagat gtaactttgggcgccgctct 11580 aacacctagt attctcattt tttatatcct attttagaat caggccgcatctctgcgacc 11640 atccatcatc caaacgtctc ttcactcatc cgaagaagca gcaatccgggttagcacgca 11700 caagctcaag cgcatcagtc agcgaaagtt cagtactgta ctgatgccatttcatatcct 11760 tccgcatcca atagattttc catctatcca gagaacgtat gtacttgattcttgctgatg 11820 gcaggatgtt tgtttcacct ggattgccct gccacacggg gcgctgttcgccgatatcta 11880 tcgtttggtc attgatgcta taaacaatat ccagttcatt gcggatatgttcaggcggcc 11940 ttatgctttc aatgaattgg tgaacttctt tttttactgc ttgatattcaaggtcattga 12000 acgccatcta tcctccttac ccaaacgtct cttcaggcca ctggttaccagctatgtgac 12060 gatgaagtca cgaacttttc agccactccc ttgcctcgat gtcatccagatggcgagatt 12120 gcttcagaat accagctaca tactccacct ttgctacttg atgataaggcaacgttatag 12180 gcctgtgatc ctggttaatg cttgtaaatt ggtattctcc atctctgtcatagccaagaa 12240 ccttaatcat gttgtgtcct tcaacggttc tgacaaacac ctcatcacccgggaatactt 12300 tggtgttagg ctcaatgagt acatattctc ctgattttat tctgggccacatgctgtctc 12360 ctttcacacg aagaccaaag gcatctggat catcgctata aattttgagccacccatcgc 12420 gctcttcggt catctcgatg gcaccatcaa caccaagaat tgcctcaccaaccacgcgca 12480 ctaacccttt ttttaatttg ccaacaaatg aaagagtatc ttcatcattcgctccattta 12540 acgaagtgcc gtgctgaagc caaacaacat caacgtttag aaatttcgcaagcgcattca 12600 ttttttcctg acgcggtaaa gactcagcat taaaccattt gctaacgcctttggacgaaa 12660 gagaaagggc acgggctata gccattcccc taccatgttc atcaagaccagcttctttac 12720 aggcttgcgc tagccgctgg gcgaattctt tgcgcacttt ttcattctgaaccatgagta 12780 cgatactaaa gcacttgcaa aaactttcag ttcaaccata atacgtactgaaagtacgaa 12840 aaaggatatt cctatgcaaa atcttgatga gccgattaaa ggtgtcggcatccctgaagt 12900 tgcgaaggct tgtggagtta gcgaaagggc tgtctataag tggctcaaaaacggcttcct 12960 ccctaagact gagttttttg ggaaaactaa atacgcatca aaaatcgaagagatttctgg 13020 tggcaaatat caagcaagcg aaatgcttga aataagcaaa aagaaccttctggctgcata 13080 agtaacaccg ctattttcac aatggacatt cgtcctacgt cgctgacaaagcgagtccca 13140 atatatctga ccaactaagg ccatatgcgt ttccacgcat acctttcaactagctattca 13200 ctattggaaa tcttaagaaa tggaacaaac aagttacagc aaactatcacagcgagaaat 13260 tgatcgcgct gaaactgatt tactcatcaa cctgtcaacg cttacccagcgcggtctggc 13320 aaagatgatt ggctgtcatg aatcgaagat aagcagaaca gactggaggtttatagcttc 13380 ggtcttgtgt gcttttggca tggcatcaga catcagtccg attagcagagcttttaagta 13440 tgcgcttgat gaaatcacaa agaaaaaatc cccggtggcc gccggggactctaagcaaat 13500 tgatatgcaa ttctgaggga attactggat caatccacag gagtcattatgacaaataca 13560 gcaaaaatac tcaacttcgg cagaggtaac tttgccgaac aggagcgtaatgtggcagat 13620 ctcgatgatg gttacgccag actatcaaat atgctgattg aggcttattcaggcgcagat 13680 ctgaccaagc gacagtttaa agtgctgctt gccattctgc gtaaaacctatgggtggaat 13740 aaaccaatgg acagaatcac cgattctcaa cttagcgaga ttacaaagttacctgtcaaa 13800 cggtgcaatg aagccaagtt agaactcgtc agaatgaata ttatcaagcagcaaggcggc 13860 atgtttggac caaataaaaa catctcagaa tggtgcatcc ctcaaaacgagggaggttcc 13920 cctaaaatga gggacatccc tcaaaacgag ggaaaatccc ctaaaacgagggataaaaca 13980 tccctcaaat taggggattg ctatccctca aaacaggggg acacaaaagacactattaca 14040 aaagaaaaaa gaaaagatta ttcgtccgag aattctggcg aatcctctgaccagccagaa 14100 aacgatcttt ctgtggttaa accggatgct gcaattcaga gcggcagcaagtggggaaca 14160 gcagaagacc tgaccgccgc agagtggatg tttgacatgg tgaagaccatcgcaccatca 14220 gccagaaaac cgaattttgc agggtgggct aacgatatcc gcctgatgcgtgaacgtgac 14280 ggacgtaacc accgcgacat gtgcgtgctg ttccgctggg catgccaggacaacttctgg 14340 tccggtaacg tgctaagtcc ggccaaactc cgcgacaagt ggacccaactcgaaatcaac 14400 cgtaacaagc aacaggctgg cgtgacagct ggaaaaccaa aactcgacctgacaaacact 14460 gactggattt acggggtgga tttatgaaaa acatcgccgc acagatggttaactttgacc 14520 gtgagcagat gcgtcggatc accaacaaca tgccggaaca gtacgacgaaaagccgcagg 14580 tacaacaggt agcgcagatc atcaacggtg tgttcagcca gttactggcaactttcccgg 14640 cgagtctggc taaccgggac cagaacgaac tgaatgaaat ccgccgccagtgggttctgg 14700 ctttccggga aaacgggatc acctcgatgg aacaggttaa cgcaggaatgcgcgtagccc 14760 gtcggcagaa tcgaccattt cttccatcac ccgggcagtt tgttgcatggtgccgggaag 14820 aagcatccgt tatcgccgga ctgccaaacg tcagcgagct ggttgatatggtttacgagt 14880 attgccggaa gcgaggcctg tatccggatg cagagtctta tccgtggaaatcgaacgcgc 14940 actactggct ggttaccaac ctgtaccaga acatgcgggc caatgcgctgactgacgcgg 15000 aattacgacg caaggctgcc gatgaactga cctgtatgac agcgcgaattaaccgtggtg 15060 agacgatacc tgaaccagta aaacaacttc ctgtcatggg cggcagacctctaaatcgtg 15120 ttcaggcgct ggcgaagatc gcagaaatta aagctaagtt cggactgaaaggagcaagtg 15180 tatgacgggc aaagaggcaa ttattcatta cctggggacg cataatagcttctgtgcgcc 15240 ggacgttgcc gcgctaacag gcgcaacagt aaccagcata aatcaggccgcagctaaaat 15300 ggcacgggca ggtcttctgg ttatcgaagg taaggtctgg cgaacggtgtattaccggtt 15360 tgctaccagg gaagaacggg aaggaaagat gagcacgaac ctgatttttaaggagtgtcg 15420 ccagagtgca gcgatgaaac gggtattggc ggtatatgga gttaaaagatgaccatctac 15480 atcactgagc taataacagg cctgctggta atcgcaggcc tttttatttgggggagaggg 15540 aagtgaacga tagctaccga cagtttgaaa actggtggtc aaaagacaaaagccagttca 15600 cgggagacga tgaattaaaa gagtttgcct gggtgatatg gcaggcatcgcgctctgcta 15660 ttgaactgga tatcgactgg cccgaatcga atgacgactt ttggaaagatggtgaagaag 15720 gtgcttatgc gatgggttat gaggatgggc gtgacaaaac ggtaattgcagtaatgaaag 15780 ccatcagggc cgcaggaatc aaagaaaaga atttcgatta agcaaatatcacttcaataa 15840 atcgctttta aggcatcaca atcgctctgt agtgaggtaa acgcgtgcaaggcatgccaa 15900 tagcagcgag aatgaaaaat gcgtcagaat gcgtttgagg aggttttaagaaatgagtac 15960 gatagctgag cttgtcaggg ctaattttcg tgaagagttg gtgcgttggtatcggtatcg 16020 ttcatcgtcc agtttgccgc ttgatgagtt gtatgagcat tcacctgccgcacgatgcta 16080 tccgcgtgac cgtgttcttc gacggttgtt caaactcaac aatgagtttcagcgcaacag 16140 aattatccgg agtctggatt ttaagtgaag gagtgagcat gagcgacctatcattaaccc 16200 agccaaagct aaaagaatgt ccgttttgcg gcggtaatgc tcgtctgtgggttgaggccg 16260 gaataaatat tgatgtgtgg ggctatgcag aatgtgacct ctgtgaagccagggggggca 16320 tgggcaccat cagttgctgc ggcggctgaa aaatggaacc ggagagcaggagatgaagca 16380 aacctttctg cttcgcaacg aagcaatcag aaataacgcc atagacgccattctctcact 16440 acccatcgac gacaagtcac cccacgaagt ccacgttaaa gaacccaagcgcagcaaagc 16500 gcagaatgac cgtatgtggc cgatgctgaa cgatgtttcg cgtcaggtgctatggcatgg 16560 tcaacggctg gcgccggaag actggaaaga cctgttcact gccctgtggcttaagaccaa 16620 aaaactggag caacgaagtg tgcctggtat cgacggtggc gttgtcatgcttggcgtgcg 16680 taccagcaaa atgcggaagg ccagctgact gagcttatcg aaatcatgttctggttcggc 16740 tcagagcgca acgtgcggtg gagtgatgac tcccggcgag agtatgaatggttacaacga 16800 aaaggtaggg ctgcatgact atcaaatcaa atacaccagc acacgacaaggactgctggc 16860 aaacgccgct ttggcttttt gatgcactgg atattgagtt tggattctggctggattcag 16920 ctgcgagcga caaaaatgct ctgtgcgctc actggctaac tgaggtcgacgacgcgctca 16980 attctgagtg ggtaagccac ggtgcaatct ggaataaccc accgtacagcaatatcaggc 17040 cgtgggtgga aaaagccgct gagcagtgca tacaacagcg acagacggtagttatgcttg 17100 taccagagga tatgtcagtc ggatggttca gcaaggctct ggagagtgttgacgaagttc 17160 gtattatcac tgatggacgg attaatttta tcgaaccatc gacagggctggagaagaagg 17220 gaaacagcaa aggttccatg ctgctgattt ggcgaccgtt catcagtcctcgacggatgt 17280 ttactaccgt atccaaagcg gcattgatgg cgatcgggca gggcgtcagaagggcggcat 17340 gaggcgacag caaagaagca tcaccgacat catctgcgaa aactgcaaataccttccaac 17400 gaaacgctcc agaaatttag ttttgagcag aataccatga tgtcagtgcaagggggagaa 17460 agtctcctca ttatctgatt cgcaatttac gtgcatattt aaatattgcacgttacaacg 17520 tgcatgtgta tgattgactt atcaatcaca acacgagata tgctcatgaaaaatgatgat 17580 gttagtggga aggccaaagg cggtaaggca cgcgcggcaa aaatgacagcagagcaaaga 17640 aaagaatcct caagaaaggc tgttgccgca aaaaaagaaa aagctttattgcccgtatct 17700 gcgaatgagg gaaagttaaa gatcggtgat gcggaattag atgtcgcggttctcgaaaat 17760 ggacggcgta tcatatcaca agcttctgtt tttaaagcat ttggccgaccacaaagaggg 17820 ggtagagcac ctcaagaaga gggggtgatc aatatgcccg cttttatggatgctgcaaac 17880 cttaaaaaat atataaatca agatgttatg ggtgtgatca ataaggtcaaatacaagacg 17940 attactggct ccgtccagga aggttatgac gcatccataa tacctcttgtctgcgatgtt 18000 tatttaaagg caagagaggc aggcgctatc accaggccaa accagttagagacagccaag 18060 aaagctgaaa ttctggtgcg ctcattagct aaagtcggaa taatagcgcttgttgatgaa 18120 gcgacggggt accagcgaga tagagaaaaa gatgcgctcg ccaaaatactcgaggccttt 18180 gtcgcaaagg aaattcaacc ttatattaca acatttcctg ctgattattatgaagagctt 18240 ttcaggttaa ggggcttaga atacccgccg gaaaatcccc gcttccggcctcagtatttt 18300 ggcgttttga caaatgatat cgtctacaag agattggcac caaacatccttgaggagctt 18360 aaaaagcaga acgtaaaggc ctcaaaaggt acaaagttgt ttcaggggctgacgccaaat 18420 attggatatc aaaaattaag agagcatctg tcatcaaccg ttacgattatgaagctatct 18480 aacgactatt cagattttat tgcaaaaatg aatcgcctgc atccaaggtttgaggatgtg 18540 aaaacagacg aactggatga ttcagacaag taacagtaac ccaccttcaggtggtttttt 18600 tgtacaaatc cttcagagta agtttacctc cttcactgca ttactactgaccattgacaa 18660 cttaacaaac ccagcttcgg ctgggttttt tattgctgaa ttttcaatgtgagaggacat 18720 gacaatgaat gagctgataa atagcaatgc catcaaaatg acaagcattgaaatcgctga 18780 gttggtggga agccaacacg gtaatgtcag aatatcaata gaacgtctggcaaagcgtgg 18840 ggtgattcaa cttccttcaa tgcaaaaagt tgaaaataaa caaacaattagccctaacaa 18900 attcacaagc gtgtatatat tcgaaggcga acaaggtaag cgaggcagcattattgtcgt 18960 cgctcagttg tcgccggaat tcaccgctcg ccttgttgac cgctggcgagaactcgaagg 19020 ggcaaccgcg aaaataccac aaaccttttc tgaggcattg cgccttgcggccgaccttga 19080 agaccagaag gctgaactgg agaaacagct tgctctcgca gcacctaaagttgagtttgc 19140 cgatcgagtt ggcgaggcca gcggaatttt gattggaaac tttgcaaaggttgttggtat 19200 tggtccaaac aaactgtttg cgtggatgcg cgatcacaaa atccttattgcttcaggtgc 19260 ccggcgcaat gtgccaatgc aggaatatat ggatcgcggc tatttcacagtgaaagaaac 19320 agcggtcaat acaaatcacg gaatacagat atcgttcacc acaaaaatcaccgggcgtgg 19380 tcaacagtgg ctgacaagaa agctgctaga taacggaatg cttaaagtaacaggggaggc 19440 tgcttaatgg ctaaaccagc gcgaaggaaa tgcaaaatct gtaaggaatggtttcacccg 19500 gcattctcaa atcagtggtg gtgctgcccg gaacacggaa ctcaattagcactcgaacga 19560 cgaagtaaag aacgcgaaaa agcggaaaaa gcagcagaga agaaacgacgacgagaggag 19620 cagaaacaga aagataaact gaagattcga aaactcgcct taaagccccgcagttactgg 19680 attaaacaag cccaacaagc cgtaaacgcc ttcatcagag aaagagaccgcgacttacca 19740 tgtatctcgt gcggaacgct cacgtctgct cagtgggatg ccggacattaccggacaact 19800 gctgcggcac ctcaactccg atttgatgaa cgcaatattc acaagcaatgcgtggtgtgc 19860 aaccagcaca aaagcggaaa tctcgttccg tatcgcgtcg aactgattaaccgcatcggg 19920 caggaagcag tagacgaaat cgaatcgaac cataaccgcc atcgctggactgtcgaagaa 19980 tgcagggcga tcaaggcgaa gtatcagcag aaacttaaag acctgcgaaacagcagaagt 20040 gaggccgcat gacgttcaca gtaaaaacca ttccagacat gctcgttgaggcatatgaaa 20100 atcagaccga ggtagccaga atactgaact gtagtcgcaa cacggtcagaaaatacactg 20160 gcgataaaga agggaaaaga cacgctatcg tcaacggtgt tcttatggttcaccgcggat 20220 ggggtaaaga tactgatgcg tgatatccgg caggttcttg agcgctggggggcatgggcg 20280 gcaaataact atgaggatgt tacatggtcg cccattgctg ccggatttaagggactgatc 20340 cccgaaaaag taaaatcacg tccacagtgc tgtgacgatg atgcgatggtgatatgcggg 20400 tgcatagccc gcctttaccg gaacaatcgc gatctgcatg acttgctggttgattattac 20460 gtgttggggg agacgttcat ggcgttggca cggaaacatg ggtgctctgacacctgtata 20520 ggtaaacgcc ttcacaaagc ggaggggatt gttgaaggca tgctgatgatgctgggagtg 20580 aggcttgaga tggatcggta tgttgagcgt gaattgccgg gagggagaacctctgtattt 20640 tatcagcgaa aaaatagttt acgatcgtaa aaatctgcat atcatgataagagtggttac 20700 attgccacgc agtcgaaccc gccgatgcgc gggttttttt gtaccccgaatcctgtgagc 20760 tatacggaaa gtacacagaa aggaaggtgc gaccgtaatt aataacaaaatcttaaaaat 20820 cgcatatagc actattagtt ttctaaatat tgtatatttt aagtattgcaggataaccct 20880 gtaacgaagt ttgcgtaaca gcattttgct ctacgagttt gccagcctcccccagtggct 20940 ggctttttta tgtccgtagc gtcaaagcag caatggcgct agggcgtcgtgcaattggcg 21000 ttgagctgga gagcgggcgt tttgagcaga cggtcaggga agttcagaatgtagtcagtc 21060 agaacggatg atattgcagg attagttacg taccgttatt atcctgcgcccggcccttta 21120 gctcagtggt gagagcgagc gactcataat cgccaggtcg ctggttcaaatccagcaagg 21180 gccaccatat cacataccgc cattagctca tcgggacaga gcgccagccttcgaagctgg 21240 ctgcgcgggg ttcgagtcct cgatggcggt ccattatctg cattatgcgttgttagctca 21300 gccggacaga gcaattgcct tctgagcaat cggtcactgg ttcgaatccagtacaacgcg 21360 ccatatttat ttaccaggct cgcttttgcg ggcctttttt atatctgcgccgggtctggt 21420 gctgattact tcagccaaaa ggaacacctg tatatgaagt gtatattatttaaatgggta 21480 ctgtgcctgt tactgggttt ttcttcggta tcctattccc gggagtttacgatagacttt 21540 tcgacccaac aaagttatgt ctcttcgtta aatagtatac ggacagagatatcgacccct 21600 cttgaacata tatctcaggg gaccacatcg gtgtctgtta ttaaccacaccccaccgggc 21660 agttattttg ctgtggatat acgagggctt gatgtctatc aggcgcgttttgaccatctt 21720 cgtctgatta ttgagcaaaa taatttatat gtggccgggt tcgttaatacggcaacaaat 21780 actttctacc gtttttcaga ttttacacat atatcagtgc ccggtgtgacaacggtttcc 21840 atgacaacgg acagcagtta taccactctg caacgtgtcg cagcgctggaacgttccgga 21900 atgcaaatca gtcgtcactc actggtttca tcatatctgg cgttaatggagttcagtggt 21960 aatacaatga ccagagatgc atccagagca gttctgcgtt ttgtcactgtcacagcagaa 22020 gccttacgct tcaggcagat acagagagaa tttcgtcagg cactgtctgaaactgctcct 22080 gtgtatacga tgacgccggg agacgtggac ctcactctga actgggggcgaatcagcaat 22140 gtgcttccgg agtatcgggg agaggatggt gtcagagtgg ggagaatatcctttaataat 22200 atatcagcga tactggggac tgtggccgtt atactgaatt gccatcatcagggggcgcgt 22260 tctgttcgcg ccgtgaatga agagagtcaa ccagaatgtc agataactggcgacaggcct 22320 gttataaaaa taaacaatac attatgggaa agtaatacag ctgcagcgtttctgaacaga 22380 aagtcacagt ttttatatac aacgggtaaa taaaggagtt aagcatgaagaagatgttta 22440 tggcggtttt atttgcatta gcttctgtta atgcaatggc ggcggattgtgctaaaggta 22500 aaattgagtt ttccaagtat aatgaggatg acacatttac agtgaaggttgacgggaaag 22560 aatactggac cagtcgctgg aatctgcaac cgttactgca aagtgctcagttgacaggaa 22620 tgactgtcac aatcaaatcc agtacctgtg aatcaggctc cggatttgctgaagtgcagt 22680 ttaataatga ctgaggcata acctgattcg tggtatgtgg gtaacaagtgtaatctgtgt 22740 cacaattcag tcagttgaca gttgcctgtc agactgagca tttgttaaaaaaatttcgca 22800 tggtgaatcc ccctgtgtgg aggggcgact ggtgaaaaat ccttgcttgtgattcattat 22860 cgacacgggt tcggtggtac caggccgaac tcaccgggag gcacccggcaccatgcagta 22920 tacagagatt aggcatatac caaggcctct catagcaggg gcctttttacatgtaaaaaa 22980 gcccgagtgg gttcgggcaa ttgcatgaga tactcgtttt aataatcgaaatcattttaa 23040 ccaggattca taaggctgcg caactgcgcg gcctttttcg tatttcgggctgtagtcttc 23100 cttctgccat tgtcctgtaa cttccggact tcagcccgct ccttattttactcacaatat 23160 tatcccggcc gggaggattc atggcattta aacactatga tgttgtcagggcggcgtcgc 23220 cgtcagacct tgcgaaacga ataactcaaa aactgaagga agggtggcagccttatggta 23280 gtgcgctgat ttcgacagct ggttatggtg cggagttcat ccagccagttgtgagtgagg 23340 ggagcatctc atcaccagag gagccaggca accgtccgac gacctcagcgccttctgttg 23400 cgccagaata ttactatgtg atcgcgcttg ctggtcagtc caatggtatgtcatacggtg 23460 agggactgcc attgccggat acattcgaca gccctgatcc acgtattaaacagttagcgc 23520 gtcgcagtac ggtgacaccg ggcggtgcag tatgcaaata taacgacatcattccggcgg 23580 accattgtct gcatgatgtg caggacatga gccgtcttaa ccatccgaaagcggacctgt 23640 caaaggggca gtacggaacc gtggggcagg ggctgcatat cgccaaaaaactgctgccgt 23700 ttataccggc gaatgcgggc attctgctgg ttccgtgctg tcgtggtggttcagcgttca 23760 ccaccggagc cgatggcaca tacagtgacg cgagtggtgc ctcggagaattcaacccgct 23820 ggggtgtgga caagccgctg tataaggacc ttatcggtcg aacaaaagcagcactgaaga 23880 agaatccgaa aaatgtgctg tttgccgtgg tgtggatgca gggggaatttgattttggcg 23940 gtacgccggt aaatcacgcc gcacagtttg gtgcgctggt tgataaattccgtgcagacc 24000 tggcggatat ggcaggccag tgcgtcggtg gctctgctgg cggtgttccctggatatgcg 24060 gggacacgac gtatttctgg aagcagaaga acgaatccac gtaccagacggtgtatggca 24120 gctataaaaa caaaacggaa aagaatatcc atttcgtacc gttcatgacggatgagaacg 24180 gggtgaatgt gccgacgaac aaaccggaag aagacccgga cattccgggtatcggatatt 24240 acggttcgaa atggcgtgac agctcagcca cctggacgtc acaggacagggcgagccatt 24300 tcagttcatg ggctcgccgc gggattattt ccgaccgtct ggcaacggcgattttgcgcc 24360 atgcgggaag agtggcgcta aacgcggggg catcatcgac agtatcagaggtgcgcccgt 24420 catcgccttc cggtgcagaa gccacaggcg tcacaacact gctctcttaccttgccagcg 24480 agtcagaggg aagcctgaaa gtacagggat ggtcagccag tggcggcagggcagaagtgg 24540 tcagcgatgc ggagggaacc ggaggtaagg cagtgaagct gaccaaggaagccggtaaaa 24600 gcagctgggt gctggagtac gccgcgggca acggtgcggc tctgttacagaaaggggggc 24660 agattcgctg ccgctttaag gtttcgggag cgctggctgc gaaccagtatgttatggcgt 24720 tttactggcc ggtatcttca ctgccacagg gcgttgccct gaccggagacggggggaata 24780 acctgctggc agcgttctac atccagacag atgcaaaaga cctgaatgtgatgtaccaca 24840 atgcgaaagt ggcgacaaac aacctgaaac tgggaacctt tggcgcatttgataacgaat 24900 ggcatacgct ggctttccgc tttgccggga ataacagcct gcaggtgacgccggttattg 24960 atggtcagga tggcacaccg ttcacgctga cgcagtcacc ggtcagtgcctttgcggcgg 25020 ataaactgca tgtgacagac attaccagag gtgcgactta cccggtactgatagacagca 25080 ttgcggtgga agtgaacagc acagacactg cggcatgata aaaaaaccgccagcgacagg 25140 aatggacgct ggcggtggtg atacctatgg agaaaaaata aaggaacgatactttcgtac 25200 tctggttttt aatgaaaaca gttcttattg tcaacaataa cggaaagaaattatgacatt 25260 tctgaaccag ttaatgctgt acttctgtac ggtggtctgt gtgctgtatctcctttcggg 25320 tgggtacagg gccatgcgtg acttctggcg cagacagatt gacaaaagggccgctgagaa 25380 aatcagcgcc agtcagtcag ccggaagcaa acccgaagag ccgctcatttagcggcaact 25440 ttcttaatca cacctttcga cgagaaaatc ccatgtcaga aattacatccctggtcactg 25500 cagaggcagt gaaggacgtc ctgcgctctg aagaagtccg gagcgcactgaaacagaaac 25560 ttcgccataa cctggaagcg cgtcttgatg cagaagtgga tgccattctggatgaactgc 25620 ttggtgtaca ggcagagcca ccgactgaag cgggagatac caccgcagagagcggtgaag 25680 ttcagcctga atcaccggtc gccgatgcga ctgaacctca acccgaatcggtcatgatgc 25740 tgtaacgggg agtcagggcc atcagtaaac agctgctggc ctttttcatgttgtgagctt 25800 ccggattgcg ggagacgggg tatgtaccag atggaaaaaa tcacaacaggtgtgtcatac 25860 accacgtcag cggtgggaac gggctactgg ttcctgcagt tgctggacagggtttccccg 25920 tctcagtggg cggcaatagg cgtgctgggg agtctgctgt ttgggctgctgacatatctg 25980 acgaacctgt atttcaagat taaagaagac cggcgtaagg cggcacggggagagtaagct 26040 gatgagcagg aaactccgct atggtttatc ggctgccgtt ctggcgctgattgccgcagg 26100 tgcttctgcg cctgaaatcc tcgaccagtt tctggatgaa aaggaaggtaaccacaccac 26160 agcataccgt gatggtgcgg gtatctggac catctgccgt ggagccacccgggtggatgg 26220 taagcctgtt attcctggca tgaagctgtc gaaggaaaaa tgcgaccgggttaacgccat 26280 tgagcgtgat aaggcgctgg catgggtgga gaaaaacatc aaagtgccgctgaccgaacc 26340 ccagaaagcg gggatcgcgt cattctgtcc gtacaacatt ggtcccggtaagtgtttccc 26400 gtcgacgttt tacagacgaa ttaatgcagg tgatcgaaaa ggtgcctgcgaagctattcg 26460 ctggtggatt aaggacggtg gcagagactg ccgtattcgt tcaaacaactgttacggtca 26520 ggtatcccgt cgtgaccagg agagcgcgct ggcgtgctgg ggaatcgacagataagaaga 26580 atattttgct gaaaaatgag gtttgcttac ctggacggat aacacgaaatcctgcaaatt 26640 ggcaaaatgt aagtgaataa agtcaaaaca gttgtttaac actcaggcaccgtaatgatg 26700 cctttgtcat ttctgcgcat ctcacgcgca tctcacaaca cagaacctttcaggatgacc 26760 cttgaggata ccggtttggc tgtcggtgcc tttctgtggg ctggattcctgtgagacaag 26820 gttcatcact aaaaggaaat aaccgatgaa tatgatggcc gtgccgtttcacggcaactc 26880 tctttatgta gttaaccata atggcgaacc atacgttccc atgaaacctgtcgttgcggg 26940 gatggggctg gcctggcaat cacagttggc taagttaaga cagcgttttgcgtcaactat 27000 aacggaaatc gttatggttg ctgaggatgg gaaacaacgc aatatggtgtccatgccact 27060 tcgaaaactt gccggctggc tacaaaccat taatcccaac aaagtaaaacccgaaatccg 27120 cgataaggtc atccggtatc aggaagagtg cgacgatgtt ctttacgagtactggacgaa 27180 gggttttgtc gttaatcccc gtaaaatgag cgtgatggaa gaactcaaccaggcttgtgc 27240 tgacatgaaa cgggataaaa acattgccag tgtgtttgct accgggctgaatgagtggaa 27300 acaggttaaa gccgcgcatg tatcaaaaat ccgtacgctg gtaaatgaagcgaatatgct 27360 gattgatttt gtcctggctg atacaggcaa agggaaaata acaaaggcggattgatgggg 27420 tggctaatga tatcagataa actcataacg ctggtgaaga gcctctgtgtacttgtcggc 27480 atttcatttt tagtcatgct ggttgccatt ttcttttcca ccgcctggcgagtcctgacg 27540 ttatcgggac tggtggggtg aaagagagat gaaccgtgtt ctgtgtgtggtgattattgt 27600 cctggcggtt ggctatggtg cgctgtggct ggcaacaaac cattaccgtgacaacgcgct 27660 cacctacaaa gcgcagcgcg ataaaaaagc cagagagctg gaacaggcgaatgccaccat 27720 tactgacatg caggtgcgcc agcgtgatgt tgctgcgctc gatgcaaaatactcgaggga 27780 gttagccgat gcgagagctg aaaatgaaac tctgcgtgct gatgttgccgctggtcgtaa 27840 gcgcctgcgg atcaacgcca cctgctccgg taccgtgcgt gaagccaccggcacctccgg 27900 cgtggataat gcaaccggcc cccgactggc agacaccgct gaacgggattatttcatcct 27960 cagagagagg ctgatcacta tgcaaaaaca actggaagga acccagaagtatattaatga 28020 gcagtgcaga tagagttgcc catatcgatg ggcaactcat gcaattattgtgagcaatac 28080 acccgcgctt ccagcggagt ataaatgcct aaagtaataa aaccgagcaatccatttacg 28140 aatgtttgct gggtttctgt tttaacaaca ttttctgtgc cgccacaaattttggctgca 28200 tcgacagttt tcttctgccc aattccagaa acgaagaaat gatgggtgatggtttccttt 28260 ggtgctactg ctgtctgttt gttttgaaca gtaaacgtct gttgagcacatcctgtaata 28320 agcagggcca gcgcagtagc gagtagcatt tttttcatgg tgttattcccgatgcttttt 28380 gaagttcgca gaatcgtatg tgtagaaaat taaacaaacc ctaaacaatgagttgaaatt 28440 tcatattgtt aatatttatt aatgtatgtc aggtgcgatg aatcgtcattgtattcccgg 28500 attaactatg tccacagccc tgacggggaa cttctctgcg ggagtgtgcggggataatca 28560 aaaacgatgc acaccgggtt ttctcatttt tcacgagatg ggagcgatttcccgcgaagc 28620 cgcctgtccg gtgcggtggt ggaagaaacc ggataaaaca accgcattgtgcaaatatcg 28680 atcaaatatg gtgctgctgt gtgaaatctg aaaaatcaca gcggtcattatgcatcagtt 28740 tttaacacag gacgtcagaa cgtgacatgg caaagctgga ctggaaaaagctggagcagg 28800 cattccgacg cgaacatgcc gaaacgggaa taacattact ggactggtgccggaagaaaa 28860 agattaatta caacaccgcc agaacccgta taaaaatggg caaaatcgatcatgaaattg 28920 atcataaaac cgatcatgaa atcgatcatg acatctcaga tgaagaaccctgcaatgacg 28980 cgggttccgg cgatgaaaaa tgtgcaaaaa actctgaaaa aaactgcgcaaattcggcag 29040 aaacgaaacg gattcgtggt tcccgacttt taccaccttc aaacgctttttctcagcgaa 29100 acacccacgc cgtaagacac cgtggatatg cgaagtatct tgaggcagataacctcatgg 29160 atgatgcgtc cgacatggtg ctgttcgatg aactggtgtt cacccgggcccgcgcacttt 29220 cagtaactaa ggcacttaaa gggatgttcg ccgacctgga agaggcaactgacgtggaaa 29280 cccgtgttgc tctttacgac aaaatactca aagctgaaca ggcccttgaccggaatattg 29340 cccgtatcga gtcaattgaa cgctcattgc tgacgctgga cgtcctggctgagacagcac 29400 caaaacttcg tgctgaccgg gaaagaatca acgccgccag agataaactcagagctgaaa 29460 ccgatattct gaccaaccag cgtcggggcg ttgttacgcc tgtcagtgacatcgtgtcat 29520 cgctgcatga aatgagtaat tcggggagac tggatgacat tccggaagaatgaaccgcga 29580 tgtgatgagc cgtcagaaat gaccgaggct gaacaacgtc tgttcatcatgactaaactg 29640 agcaatccct ggtggcggct caatcatctc tacaaaatac agaacgaaaaaggtgaactg 29700 gtcaccttca gaatgcgacc ggcgcagcgc cagttgttcc ggagcatgcacaataaaaat 29760 attatcctga aagcgcgcca gctgggattt tccacagcca ttgatatttatcttctcgac 29820 caggcattat tcattccgca tctcaaatgc gggatcgtcg ctcaggataaacaggctgcc 29880 agtgaaattt tccgcacaaa aattgctgta ccgtttgatc atctccctgactggctgaga 29940 gcctcattca ccatcgttga acgtcgtagc ggtgccagcg gtggctatatcctgtttggt 30000 cacggctcga gtatccaggt ggcaacctca ttccgttcag gtacggtgcagcgcctgcat 30060 atctcagagc acggcaaaat ttgcgcgaaa tatccggcta aggcgaaagaactgcgaacc 30120 ggtacgctta atgccgtctc tgatgaatgc attatttttg atgagtccactgctgaaggc 30180 gtgggtggtg atttttacga gatgagtaac cgagcacagg agatcactgcatcaggctta 30240 ttgctgacgg cacaggatta taaattccat ttttacgcct ggtggcaggatcctaaatac 30300 agcgccagag tgccggaaag cgggctgaag ctgtcacggg aaaaaatgacgtatttttct 30360 gcggttgaga aggcaatgaa catcacgctt actgatgaac agaagcagtggtacatcaat 30420 aaggaaactg aacagcgtga ggaaatgaag caggagtttc cctcaacgccacaggaggcg 30480 tttctgacgt ccggacgacg tgtgttcagt gccgaaagta cgttgcaggcagaatcattc 30540 tgttcgccac cgatgattgt ttatgacatt gaacctgtta caggagcgaagactaaagct 30600 cagtctctgc gtgaaggaaa taaaaacgag ttgcagcgga cgctgatgaattatctgctg 30660 gtatgggaac tgccggatcc ggatgaagag tatgtttgtg gggcagatactgccgaaggg 30720 ctggagcacg gagaccgctc atcgctggat gttgtcaaac gcagtaatggcgagcaggtg 30780 gctcactggt tcgggcatct cgatgctgaa ctttttgctc atctcatttcgcaggtctgt 30840 cgtatgtata acaacgcgtt tgtggggccg gagcgtaata atcacggacatgcagttatc 30900 ctgaaactcc gggaactcta tccgacacgt tatatctaca acgaacagcatcttgaccag 30960 gcatatgacg acgatacgcc ccgccttggc tggctgacaa cccgtcagagcaaacctgtt 31020 ctgaccgaag gaatgaaaac gcttctgaat aatggaatat cagggatccgctggtcaggc 31080 acattatcgg aaatgaacac ctacgtttat gacgcgaaag gctccatgaatgcacaggaa 31140 ggctgctttg atgatcagct catgagctac atgattgccc aggagatgcgcgccagaatg 31200 ccggtgaggg taaaacagaa aacggataaa cgcagaacca cacactggatggctcactga 31260 tgaaaaatga aactaacacc atggcgacga aaaacgacaa tggagccacgccgcgttttt 31320 ctcagcgcca gttacaggcg ctttgttctg atattgacag ccagcctaaatggcgtgatg 31380 ccgcaaacaa ggcctgtgcg tattacgatg gcgatcagtt gccaccggaagttcttcagg 31440 tactgaaaga tcgcggtcag ccgatgacta tccataacct catcgcgcctaccgtcgatg 31500 gcgttctggg aatggaggcc aaaacacgga ctgatctggt ggtgatgtcagacgagccag 31560 atgatgaaac tgaaaaactg gctgaagcta ttaatgctga atttgccgatgcatgccgcc 31620 ttggcaatat gaataaagcc cgctctgatg cctatgcgga acaaatcaaggcgggcctca 31680 gttgggtgga ggtcagacga aacagcgatc cgttcgggcc tgaatttaaggtgtctactg 31740 tcagccggaa tgaggttttc tgggactggc tgagccggga ggctgatttaagtgactgcc 31800 gatggctgat gcgtcgccgc tggatggata ccgatgaggc aaaagctacattcccgggaa 31860 tggctcaggt tatcgattat gccattgatg actggcgtgg ttttgtcgataccacggtta 31920 ctgaaggcca gcccagtccg ttgatgagtg catgggaaga gtatcagtcatgggatcgac 31980 agcagaacga atggcttcag cgtgaacgcc gtcgtgtgct gcttcaggtggtttattacc 32040 gtacattcga gcgtcttccg gtgattgaac tcagtaatgg acgggtggtggcctttgata 32100 aaaataatct gatgcaggcg gtagctgtgg catccgggcg ggttcaggtgaaagtcgggc 32160 gggtaagccg tattcgtgaa gcctggtttg tcgggccaca ctttattgtggatcgcccct 32220 gtagtgctcc gcaggggatg tttccgctgg ttcctttctg gggataccgaaaggataaaa 32280 ccggggagcc atacgggcta atttcccgcg ccattccggc acaggatgaggtgaattttc 32340 gtcgtatcaa gctgacctgg ttgcttcagg ccaaacgcgt gattatggacgaggatgcca 32400 cccagttgtc agacaacgac ctgatggagc agatcgaacg tccggatggcattattaaac 32460 tgaatccggt ccgaaaaaat cagaaaagtg tcgcagatgt ttttcgggttgagcaggatt 32520 ttcaggttgc cagccagcag tttcaggtca tgcaggaatc ggaaaaacttatccaggata 32580 ccatgggagt gtattccgca tttctcgggc aggattcagg tgcgacgtcaggcgtggcta 32640 tcagtaacct ggtggagcag ggggccacaa cccttgcgga aatcaacgataactaccagt 32700 ttgcctgcca gcaggtggga agactgttgc tggcttatct tctcgatgacctgaaaaaac 32760 gccgtaatca tgcagtggtg attaatcgcg atgatcgcca gcgtcgccagaccattgtcc 32820 tcaatgctga aggtgataat ggtgaactga ccaatgatat ttcaaggttaaatacacata 32880 ttgcgctggc gcctgttcag cagacaccgg cgtttaaggc acagcttgcacagagaatgt 32940 cagaggttat tcaggggctg ccgcctcagg tgcaggctgt tgtgctcgacctgtgggtta 33000 atcttctgga tgtgccgcag aaacaggagt ttgttgagcg tattcgtgctgcgctgggga 33060 cgccaaaatc accggatgaa atgacgccgg aagaacagga agtagcggcacaacaacagg 33120 cacttcagca acaacaggca gaactccaga tgcgcgagat ggctggcagagtggcaaaac 33180 tggaagctga cgccgccagg gcacatgcag ctgcacaacg ggataatgccagtgcacagc 33240 gggaagtcgc cctgacacag gggcagcgtt atgtggatgc gcttaaccaggcacatacgg 33300 cagaaatcat taccggcgta cagaatatgg aacaggagca ggacgttcttcagcaacaga 33360 tgctgtatac gttacaacag cggatgaatg aaatgtcgct ctgaaaactctggcttcaac 33420 tgaaccccgt catcgtacgg ggttttttgt ttccggaggt aagcgttccgggagcggtgc 33480 gcttattcgc gggggcagcg ataagcctta tttactcaac cattcggatctgtccgataa 33540 acagaccatg cggagttatt tatggatttt gaatttacgg gtgaagaaaccccggaacaa 33600 ctggaaaaaa tgctggaagg acttggggat gtggatattg acagtcacgcacaggacgtc 33660 gtgacggaag ataccacgga aaaacatgcg gatgaggaag cacagactcagacgggcgat 33720 aacaatgtgg caccgacgcc ggatgccagt gtggagcaga cgcaggacgtgaaggagccg 33780 gaagcgaagg gggtgctcac ccgcgacggt aaacacgtca ttccctatgaagtccttgag 33840 gctgaacgtt ccggtaagca acgggccgaa caggaagccg cacttcttcgtgggcagata 33900 gctgaagaaa aacgcagggt ggaactgctg acgtctcaga tccaccaggccggtatgaag 33960 cccacaccgt taccggaaaa cgaaaaaatt tctgatgagc agattgcccgtatcagggag 34020 atgtatccgg aaattggtga cgcggtggct tcgctcatcc gtaaaaataactatctccag 34080 tcccgtgttc agcaatcagc acagcaggca gaaggtaatg gtggtgaggatttatcaccg 34140 gttcttgatg cgatgaatgc cgtgccggtg ctgaaaacgt ggcaggagtccgatccagat 34200 cgcttctcgg ttgctgtatc catcgacggg aagctccaga atgaccccgcatggaaagac 34260 aaaacgctca ctgaacgttt cgctgaagtg gcccgtcgta cgcaggttgctttcggtgaa 34320 gtcagtgagt cgtctgctga caacaaggca gacaaaacgg atatccggaaaacggcggaa 34380 gagaaagtga agaccgctga acaggagcag gcagtacctg cttccccgtcagatttaggc 34440 accacggctt ccgtcggaac cggtgataat tttgaacggt tacttggcgcttctcattca 34500 gaggcagagg cgattatgcg cggtatgacg aatgctgaaa tagacgcgcttctggagaag 34560 ctcgggtaac ttactgaagg agaactgaag taatgacgac tgtaacatcagcccaggcga 34620 ataagctgta tcaggtggcg ctttttaccg ctgccaaccg caaccgctcgatggtaaata 34680 tcctcactga acagcaggaa gcgccaaaag cggtttcgcc ggacaagaaaagcacgaagc 34740 agaccagcgc gggtgcgccg gttgtccgta tcacagacct taacaaacaggccggtgatg 34800 aagtgacctt cagcatcatg cacaaactct caaaacgtcc gacgatgggagatgagcgtg 34860 ttgaaggtcg tggtgaggat ctcagccatg ctgacttctc cctgaaaatcaatcagggac 34920 gtcacctggt ggatgcaggc ggacgtatga gtcagcagcg cacgaagtttaacctggcat 34980 cctcagccag aacgcttctg gggacgtact ttaatgacct gcaggaccagtgtgcgatag 35040 tgcatcttgc tggagctcgt ggtgattttg ttgctgacga cactattctgccgacagcgg 35100 agcaccctga attcaaaaaa atcatgatca acgatgtact gcctccgacacatgaccgtc 35160 acttttttgg cggtgatgcg acaagctttg agcagattga agcggcagatattttttcta 35220 ttggcctggt ggacaatctc tccctgttca ttgacgaaat ggcgcatccgttacagccgg 35280 ttcgtctgtc cggtgatgaa cttcacggag aagatccata ttacgtcctgtacgtcacgc 35340 cgcgtcagtg gaatgactgg tacacctcga cgtccggtaa ggactggaaccagatgatgg 35400 ttcgtgccgt gaaccgtgca aaaggtttta atcatccgct gttcaaaggtgaatgtgcga 35460 tgtggcgcaa tatcctggtt cgtaagtatg cgggtatgcc gatccgtttctatcaggggt 35520 caaaggttct ggtatcagag aataacctga cggcaaccac gaaagaggtcgctgctgcaa 35580 ccaatattga ccgcgccatg ttactggggg ctcaggcgct ggcaaatgcttacggtcaga 35640 aggcgggcgg tcacttcaac atggttgaga agaaaacgga tatggataaccgtactgaga 35700 tagcaatcag ctggatcaac ggtctgaaaa aaatccgttt ccccgagaagagcggcaaga 35760 tgcaggatca cggcgtgatt gccgttgata cagcagtgaa gctctgattttttcctttcc 35820 ctatgccggg ttttcgcccg gctttttcag gagtcattaa ttatggcaaagactatcctt 35880 gccccgtcac tgagtgaacg ggtctatacg ggtacgcacg gtaatgagtcggtggcagaa 35940 ggcgtattta cggtgaatgc tgcggaagcg gacagtgtta ttcatcttctctcactgcca 36000 gtgggcatcc gtatcaactc actccaactg gtttcaacgg gtggtctgggtactgcaacc 36060 gtcagcatta agtccggtga gcatgctctc atcgataaca gcgaagctgtttctgcaaaa 36120 tttgccagat atgtgccagt ggagccgtac accacacagc gtgacggggagctggttact 36180 gtcaccatta agactgccgc tgcaaccggc accctgaatg ttctgctgcgttataccgtg 36240 gtgggatact gattaaaacc ttccggcccg cgtcatgcgg gctttttatccggggaatta 36300 tatgagtgag aaaattgccg ttgtctatat cggcccaaaa cccgtgaaaaaggacaccat 36360 taccggaagt cgcacgctgt tcccacgtct tgagccggtg catgttgacagcgcgatggc 36420 ctggcaactg ctggggtttc cggatgtctg ggttcgtcat gaagagcttgatgatgttct 36480 gaaaaagcaa caacagaatg agcagttgcg gcaggcacag caggcgcaggaaagagtgct 36540 tgctgcgctg gcagaagcgg agaacagttt tgttgtttct gttaacgggcaggaggtgga 36600 tttaagtaag ctcacctcag cacggctggc gacgctgtgt gaggcagaagagctggatat 36660 tcacaaagac ccgaaagaaa cggctgaggc attccgtatc cgggtgcgtgaggcatttcg 36720 ccgtcgtgtt gcggagactg aacagcatgg cggaactgag tgattttttaccgtatgtcc 36780 gtcgtcatat cagcggtcca ctgaacatta tgatgacgga tgctctgtcaatggctgccg 36840 tggcattcag ccgccagtcg ttggtgtgcc gtcgggaggt tactgttgtaccagtagcag 36900 gaaaagaaat cgtgcttccg tatgacaaag atgatgagga gtgcgttcatatcatccgta 36960 tctctgacga taatcatgag ctttttgtcg gtcgggatgt ggatatcagctccggacgct 37020 ccctgcgatt tgcctgttct cccggtgagg tgagcgtgct ttatgccgtcgctccgaaag 37080 ccggacgcag ccagataccg gatgaactcc tcacatggcc tgaagaagtggctgcggggg 37140 cacttgagcg gttgttcatg cagactggtg tttcatggtc agatccgttacgcgcacagt 37200 atttttctgt gcagttttct gaggggatcc gtcgggcata tcgtcatacactggcgacaa 37260 gcccgtactc ttcataccgc aaccctgtac gcaggcagag gtttttctgatgacgacgat 37320 tacagaaatc atcggacgtg tgaatacaca actggttgac ccgatgatggttcgctggcc 37380 cctgcaggaa ttgtgcgatt attacaatga tgctgtgagg gcagtgattctggcgagacc 37440 ggatgctggc gcaagcctgg aaacaataag ttgtgttcct ggcgcccgtcaggttttgcc 37500 cgatggtgta atacaacttc ttgacgtgat atgcctcagt gacggtagtgcagtcagacc 37560 attatcccgg gaggtgctgg atgcgcagta tcctgagtgg cccacaatgaagggtattcc 37620 tgaatgtttt atcagcaacg acctgtcccc gcgcgtattc tggctgtttcctgctcctga 37680 caaagagata agtattgatg cagtggtaag ccggataccg gaggcagtgtatgttctgac 37740 gcaggacgat gatacgccag ttccactgga agaggcttat gttaacccactggtggagtg 37800 gatgttgttt cgcgctttca gtaaggatgc tgccggtggc gcagaatcggggctggctgc 37860 gcagcattat cagagttttg ttgagcaact tgggatcaaa cagggggcagacagtgcatt 37920 gtatgcccgt aaaaaagtgt ttaacggagg tggagtgtga gtgttgttgtttcggggacg 37980 ctgaaatctc ctgatggtga ggcgatatca ggagcaaata ttaccctgacggcgctgaca 38040 gtttcaccgg atgcgctcag cggcaccagt gcgtcggcag tgacccgtgaaggtggatat 38100 tacggaatga cgatggatcc gggggagtat gcggtttcgg tgacggtgaaagggaagact 38160 gctgtctacg gacgtgtgcg tattgagggg accgaaagta cggtgacgctcaatatgctg 38220 ttacgccgca gtcttgttga ggttagcata cccggagaac tgctgacagatttccggcag 38280 atacagaata atgtggctga tgaccttgcc actattcgtc gcctgaatgaagacacggcg 38340 acaaaaaaca ctcaggccac acagtcaaaa gaaagtgcag cagccagtgcgaagagtgca 38400 tctgacagtg caaagacggc aaccagcagg gcggctgaag ccggacaaaaagcgactgat 38460 gccactgagg ctgcgacccg tgcagtcaca gcagcgggga atgcagaggaaagctcgacc 38520 cgtgccggag agtctgaaaa agccgccgga gctgatgcag aaaaagccagacagcatgct 38580 gaaaaggcca ggctggcgca ggagagcgcc ggagagatcc ttaagcgggcagaggctgcc 38640 actgtcagtg ctgaagaggc cagacgtatg gctgagaatg cacgggggccccgggggcct 38700 cagggagaaa ctggtccgaa gggggatgtc ggtcctaaag gcgaaacaggtccagtgggc 38760 cctcaagggc ccgcagggcc gaaaggtgag cgtggtgacg ttggtgctcagggggctgta 38820 gggcctgctg gtccgcgtgg tgagaagggc gaacaggggg agcgaggaccgcagggaata 38880 ccaggcctga agggggatac cggagagcgg gggcctaaag gggaccagggggatatgggg 38940 ccaaaaggcg agaaaggtga tccgggaggt cctgcaggcc cgcaaggtcctaaaggcgaa 39000 cgaggagaag ccggaccaca gggaccgatg ggagcacgag gtgagcgtggggagactggc 39060 ccccgaggtg aacctggtcc tgcaggtccg agaggcgaac gaggagagaccggacctcag 39120 ggacctcgtg gagagccagg tccggcaggc agcgctgcaa atgtggctgatgcaacgacg 39180 gcacagaagg gaattgtgca gttaagcagc gcaacggaca gtgatgatgaaacgaaggct 39240 gccaccccga aagcggtgaa agcggcaatg gatgtggcaa atgaagcgaaaacaaaggca 39300 gaagaggctg cagcaggagg tggtgttccc ggtccgaaag gagataaaggggacacgggg 39360 ccagcaggtc cggctgggcc gaagggtgat aagggagagc gcggtgacaccggccctgtc 39420 ggggcaaccg gcgaacgggg accggcaggt gatgctggtc cggcaggcccgcaggggccg 39480 aaaggtgaca ggggagagcg gggagagacc ggtctgacgg gaaatgcaggtccacagggt 39540 ccaaagggag ataccggtgc ggcaggcccg gcaggcccac agggaccgaaaggagaaaca 39600 ggtgcggcag gcccggtggg ggcgaccgga cctcaggggc cgaagggcgacccgggggag 39660 acgcaaatac ggttccgtct ggggccggga aacattattg agacaaacagccatggctgg 39720 ttcccggata cagatggcgc actcatcacc ggactgacct ttcttgaccccaaagatgcc 39780 acacgggttc agggtttttt tcagcatttg caggtcaggt ttggtgacgggccgtggcag 39840 gatgtcaagg ggctggatga agtgggcagt gatacaggca gaacaggagaatgacatgaa 39900 tattttgaga aagcttatgc agagtctgtg cggttgcgga aagcatgatgactgtgaaaa 39960 cgggcagtcg cttacagcac aactgcgact gggaccggca gacattctggagtcagatga 40020 gaatggcatt attccggagc aggacagggt aatcacacag gtggtgatactggatgcaga 40080 taaaaagcag atacagtgtg tggtaagacc gctgcaaatc ctgcgtgctgacgggacgtg 40140 ggaaaatatt ggcgggatga aatagccgac agcttcacaa aaaccggagcccggctccgg 40200 tttttgttgt catgtatagg ggggggttat tagagagtga agtaataaacatgttaatac 40260 gatggagtga aggatgccgt gtaattctgg ttcaagagtt ttttatgccggaaaatcgca 40320 ggattattct ggatagtaaa gaatcctggt taataatctg tgatagtcagttgggccatt 40380 taatgcgcag tatgtatcag ggacgccgtt ttattcagct gaatctggaaaaattgaaag 40440 gggtacatga tgtcgccttg ccagtgaaat gggaattcac acgaagacagtgaatagctt 40500 tctgtatacg ggaatggcga aaaatggact gtatggtgtg agtgtgaaacatcttgcgtg 40560 tgcggagtga tgcttctcgt tgctacagcg gcaatgataa tgcagtgaaaaaaggggagc 40620 aatatgctcc cccaaaccga aagaaaattg caataatcaa tgaagttatttagtcatcat 40680 cagaatgtca tgcaaggcat tttgtttcag tgatgccgat cgcgattttagcgaattcca 40740 tcataaatcc cctgattttt aagcctgaag cagtcaaagg aatttctatgccctatatcg 40800 atatcaccac gatgcgtggg atgatgccgc gcgttgtgac atccatgctgcccgagcatt 40860 ccgctgtact ggcggaggac tgccatttcc ggtttggtgt tattacaccagaacgtcaga 40920 tatccggggt tgagaaaaca ttcacaatta agccaaaaac aatttttcattaccgtgacg 40980 atttctggtt tgcatggccg gatgtggtgg atgtgatccg cagtccgatcgctcaggacc 41040 cccacgggcg tatttactac actgacgggc gttttcctaa agtgacggatgcgactattg 41100 ccacaaaagg ggacgggaat cacccgacat catcgtatcg tctggggatccccgcgccga 41160 cgacagctcc tgtctgtact gttcagcagg gcggtgatgt ttctgacgataacccgaatg 41220 atgatgaaac ccggttttat acggaaacct ttgtctcaga ttatggtgaagaaggtccgc 41280 caggtccggc gtctctggag gtaacactcc gtactccggg aactgcggtacaactgacgc 41340 tggctccggt gccattgcag aatgccagta ttaaacgtcg ccggatttatcgctctgcat 41400 caggtggagg ggaggcggat tttttacttg tggctgaact ggatgcatccgtgctcagtt 41460 acacggacaa aataccggcg aaaaaccttg ggccttccct ggcgacatgggattacctgc 41520 cgccgccaga gaatatgaca ggcctttgcc tgatggctaa cggtattgccgccgggtttg 41580 ccggtaatga agtgatgttt tcggaagcgt atctgccgta tgcatggccggaagtgaatc 41640 gtcacacgac ggcagaagat attgtagcta tctgtccgct gggaacgtcactggtggtgg 41700 cgacaaaggg ggagccttat ttgttcagtg gggtatcacc gtccacaatttctggttcca 41760 aaatcccttc aatgcaggcg tgtctgagca ggcggagtat ggttgcgatggagggttttg 41820 tgctgtatgc aggaacaaat ggcctggtgt ctgttgatgc aaacggtaatgtcgcgctgg 41880 cgacggaaca gattgtttca ccggaacagt ggcagagtca gtttaatccggcctccattg 41940 tggcttatcc ctggcgtggt gaatacattg cctgttacac gaaaccggatggtaagcagg 42000 atgtgtttgt attcagtccg gtgaacatgg atatccgtta tctcagtacaccgtttgact 42060 gcgcatgggt tgatctcgcg aaagatatga tgcgcgtggt gacaggagacaaaatgtcag 42120 tgcttgccgg gggggctctg ccctccacga taaggtggca ttcaaaaattttttcattac 42180 ctgaaagaac ctctttttcc tgtatcaggg tgaaatctcc ggcgcctgagcgggtgggga 42240 tcaccattat ggctgatgat gttcctgtga ttcattttgc gccgggtacgtttaagggaa 42300 gtgtggtgag acttccggca gcaaccgggc aaaactggca ggtgatggtatccggattcg 42360 ggcaggtgga acgaataacc ctgagtacat caatgtcgga gatgccggtatgaccagaaa 42420 accgtggcgt gcggggaagg atttatccac tgttgtggag aatatggaaattggcaccgg 42480 gcagcgtggt gacggacgtc acgcatttgt gacccgtgag gaactggttggtcttaaact 42540 cgcccggcgt cgaacatcgg gtggtgcctc atatgcactg aatccgggtattgagattga 42600 cagtacttta atgactgttg attttcccac aaaaccgctg aattttaaggcgaccggtgg 42660 atttggctcg gttcttcttg aatgggatat gcctaattat cgcggacattcactgactga 42720 aatctggcgg ggtacggagg atgaccttgc tgatgcagtg ctggttgccacgacgccggg 42780 gcaggtttac ggcgatccgg ttgaccctgg ctggtcggga ttttactggatacgttttgt 42840 taacgcggca ggagtgaaag gtccatggaa tgctgaaaaa ggcactcaggcacaaacaca 42900 gatcggcgtg aaggccatca ttgaccagat ccgcgatgag gctgcaaagtcgccggttgt 42960 gtccgagctg cgtaaagaaa taaaaaacgc gcaggggcag gctgtaaaggatgctgcaat 43020 taagacaacc gaagttgtag ggactctcag ggaagaaacg acaagaacgattggtggtat 43080 tgaaacccgc attagcacac tggattcgtc aaccagtgaa tcgcttaatgaggtcgacaa 43140 gcgcatcact aaattggata aagaaggcgg tgaagctttt cttgcaatgtggtcaaaaaa 43200 agcgggagtt gatggtatca ctgcggggat cgggattgtc gccggaaaagacagtgaagg 43260 caggcctgta agtcaggttg caatttctgc gtcgcagttg tttgtctttgacccgaataa 43320 tccggataac acagcctatc cgtttgcggt atcaggtggc aaggtagtgatcccgaaagc 43380 gatgatttat gacgcggtga ttgaaacact ggtgtcgcgg aaggttgtggcggatgaggt 43440 aaaagccggg gtaagtatca cttcgccagt tatccggagt gccgttattcagaacggaaa 43500 ctttcaggtt gattctcagg gtaacctgaa tattggaggc cttttcagtgttacgtcaca 43560 agggcaactg acaattcgtt actctaatca gaatgtagga ctggtgatccgcaatgataa 43620 aattgaggtt tatgaccaga atggacgact ggctgttcgc ataggcagattacgctgatc 43680 aggaggtgag tattggaata cggttttgcc atttataaca gaaataacgttaatgttacg 43740 ggcgtgctga ctccagtatt tttcctggac agatttacag cggagtctggctcaaagacg 43800 tacactaata aacccgacgg gaaatcattg caggctgtat gttgtttatttccctggaat 43860 aatgtatttg cggatcggaa agtaccgaag ataaccatta atgacaatacggtgacgtgg 43920 tcgaatcttg agcagggtat gggatcttat atttatacat tctggggataagtgttatgt 43980 acggtttgag cattatgaag ccggatggca gcgtatggat aagtccaggttttacgccgc 44040 agtgtctgat caacaaaggc accataccgg cgactgaaaa gtctttttttaaaacatcaa 44100 tcccgtcagg caaaagttgt tttttcttta tcagaacaga gaagaaggccgatgtcatgt 44160 acacgcatga acagattgat ggatatcatg cactaaggct tcatgtaattgtcaggggaa 44220 cgaaccctgg tgttacgacg gtttatgctt tcgcgaatat ggttactccaccttctgagt 44280 atggtatcgc catgtataac ccggacggtg agatgattta tcatggcgaaatgatgctgc 44340 ttgacgcgaa gttaatacct gttgatatca aatttgaaaa ggaccttggatatccatgcg 44400 caatcatgcc tgcactggtc gggtattata actggaaaag aactccttatgatcgaccga 44460 tttataccac atccactggt gctacaggaa ataaaatata ttcctgtgagcattattccg 44520 gtggtgcaac atgggatatt cgaaagccgt atatagataa ggtcctggttattaatacat 44580 cagtatatga ttagttgaag cgagtcttta atattcattt aaaatgtctaaaaagatgta 44640 ttattaaaaa gtttagcgtg ttatctgaat acaggatatc ttaaatgaagagtatagcaa 44700 cactggttgt gtgtgcaatc tccgggattg cctgtgtaaa tttatctgcacatgcagcag 44760 aaggagagca tacaatttct ctggggtatg cgcactttca gtttccgggactgaaggatt 44820 ttgtaaagga tgcgactgct cataacaggg agactttcag tcatttcgtcaacagaaact 44880 acttttcttc attgggcgaa tatacagatg gtcgggtcag tggatatgaaggcaaggata 44940 aaaatccaca gggcattaat atcaggtatc gctacgagat aacggatgattttggcgtta 45000 tcacctcttt tacatggacg cgttctctca ctaactcaca gacatttattgatgtgcagt 45060 cagccgatca taccaggaag attaagaatc cggcagcttc tgccagaacggatatcaggg 45120 cgaattactg gagtctgtta gcggggcctt catggcgggt taatcagtacatgagtttat 45180 atgcgatggc agggatgggc gttgctaaag ttagcgctga cctgaaaattaaggacaata 45240 ttaacagtag tggcggattt tctgaaagca acagcacgaa aaaaacctcccttgcgtggg 45300 ctgcaggtgc acagtttaac ctgaatgaga gtgttacact ggatgtggcttacgaaggtt 45360 ccggctctgg cgactggcgc acgagtggcg ttactgctgg cattggcctgaaattctgac 45420 ctgtatccgg taaccgttta ctacccgctg tgatggcggg ttttttattgcccgtacagg 45480 gcaaaaaccg taaattatgc gtgggtgcct ttcggctgat ggctggagggtgaacctgaa 45540 ggcctgatgt ggaaaggccc cgagtcaact taacgttaac ccgaggccctaacacttcgt 45600 accttaagca agtagaaggt tagcgcctct ctgtaaaagg agtcaagcgctatgtcgcaa 45660 aaatcgctta tcaccgtcac aatttgcatg acggttatct tcaccatctggatgttgcac 45720 ggttcactgt gtgagttccg gctgaatttg tggggagcgg agtttgcggcgttcttacag 45780 tgtaagcagt aggaaaaccg cgacggggac gagagtcccc gtcaactggttgctgaggtt 45840 cagccgatat ggcacccgtt tcaggtgaga gaatgaacga taaaattctctggtatatgc 45900 agcgtgttgt gagaaattcc cgcaaccctg aatttatgaa tgaagttaaagacgcctgcc 45960 ttaaaaagca ggcgttttgt tttgaggcac ctgatggctt tttggtgctgcgttctgtgc 46020 tcagtgctga tggtatccct tatgttctgg tgttgctggg cgtgtgtacggggagtaaca 46080 gcgttgagcg ttacctgccg gaggtgaaga cattaaccca tctggctggcggacgctggg 46140 ctgagttcca tacggcaagg cggggattta tccggctggg aaaacgactgggctttgagc 46200 gaatgccgga tgatgaggat ggcttcatgg tgttcaggat agcggtctgactgctacagt 46260 tttcatcatt gtgtttaaac caacattgta attcacattc tgaccctgctccggcagggt 46320 tttttgttat ccagggggcc attatgggtg gaagtaaagg cggtggtgataccaaagtaa 46380 aaccaacagc agcgcaaata gcacaggaag aagtggcctg gaaagggtggcaggattaca 46440 aaaatatcct ccgcccggct gaagataact tcatggaaaa ggtcgatgacctgaacagtg 46500 agcagcagta cgacaatatt gctggcacca caaatctggg gtatcagaaacagtttggcg 46560 aagcacggaa ggagcttgcg ggtaatcttg ctcagtccgg cgttgacccatccagtggtc 46620 gttttaatgc ggtaatgaat gcgaaccaga gtgaccaggt aaccgggcagattgacacaa 46680 ccacacgggg gcaggtatcg caggcagata agtatgttgc cgggctacaggatgttgctg 46740 ctctcggttc tggtcagaag gcggatgcgt tacagagttt taactctctggcagacagca 46800 gtctggcaaa agctaaatcg gatgcacagg cggcgtttac gaaacagcaggggcgagcct 46860 ctcttgttgg cgctggtctg ggtgcggcag gtgcatatgc gatgcataaggctggcggta 46920 gcggaggaag tggcggtgct aaaacacctg gcaccggcgc taatgccattcagcatcagg 46980 ctcagaactg gagactgtaa ttatggagta tggcaaatac gaaactctcgcaagggctgg 47040 ttattcagga gcagaccgcc cacagggtga ctggcagacg tcagcagcgctgacacgcca 47100 acaatacgac gactggcgaa ccagatattt gccccgcgtg gcaaggctggctgaccttgg 47160 ggagaacaac agtctgatga atgcacagct tgcacgggtg ggaggccttgccacttccag 47220 tctccgtaca gcgcagatgg cgcaggataa ccagatggcg agatacggggtaaaccgccc 47280 ggataatccc gacagtaata cgctggggtt acgtaatgcc ctggcaattgctggcgcgaa 47340 aaatggtatc cgtgaagccg aacaggatcg ccagatgaat attctgacgggggcttctgc 47400 accggcaaga cagaaactga gtgttggcgg ccaactggtg gcagcgtaagggggcaatat 47460 gggatacggt ttactggata ttgcaaatca gtcgcggcgt gaggcattacagggaataag 47520 tgacgcagac agacgacgtg aagaaattga ggctgcgaac aaacagatggcggcgcaaca 47580 gaaagcgcag aacaagcaga atatcggtac gggcattggt acgggggcggctattggcgc 47640 atccgttggt ggtcctgttg gtgctgttgc tggagcagta attggcggcattgctggttc 47700 tttgttttaa ggagtggtga atgagcggat ttgcacaggg gttacttgccggattcagca 47760 ccgttgacca ggcaatgacc cgtcgtaagg agcttggtct gcgtgaagcacagcttgccc 47820 ggcaacagaa aaataacgag cgcgattttg agtttgcgca gtctcagtttgaacataata 47880 aaaacgttga tcagcggaac tttgattaca gagccaaagt tgacgaccgtaattatgcac 47940 tgaaggagag ggagtttaac gctaaccaga attaccggaa tgcgtcactgggtatggagc 48000 agcagcgact ccagttgcag aaatacaacc agcgacggct tgagtataacgatatgattg 48060 cccatagcca gccactaatg gaagcgcttg gaaaagcaat tgaggctggcgatcaggagg 48120 ctgcaacgcg tctgttcggg cagctgccaa agggacatcc attaattcttatgtcaaacg 48180 aaggctatgc agcgaaagcg ggtcaggccg tgatcaacct gcaaaaaatctttggtgata 48240 agccggacat ggcgatcgat tcgctgaata ccccggaaaa tctcgatgtgctttccggcg 48300 tgtttgcccc ggaactacaa cagcgtattg gcatgcctga ttcaaccggggacaaaacga 48360 taaaagaggc caggattggc agtatcgtac cagcgcagca ggaagggtacgtacttattg 48420 gccttgatct cacatacagc gatggctcca ccgcgcataa acctgtaacagaatacggca 48480 gtgcgcaccc tgatgatcaa accgtgctgg cgatacccgt tgataaggctatcgctcagg 48540 tcagggatcg cagcaaattt gcagagatat cgaaaaatta tggttattttatgccgaagc 48600 agcagggact ttctctgaaa gagcttcaga agggggccag caacgtagcggcggacgcga 48660 tcaagaatgg cggtaatgct caggctgcgg tggatgaata ttatgctgcgactggttcac 48720 aaccgcatca acagaaaatt cagcaacaaa aacttcagca acaggttatcaactgggcgg 48780 gagatgatcc tgacaagctg tcatttgcca gaaatgtagc ggcccgtcagcctgaaatgc 48840 tggaacctca gaatcagaaa ttgctggaga acgggtatgc gaattttctccgtattcaaa 48900 aggccagggg ggaacaggcc agagatgaaa gtgcttcatc tgcatctcagtttatccgtg 48960 gactgaaaca gaattacgcc cagtaattca cgatattcca ttaataccatttcctgatgc 49020 ccggccattg tgccgggttt ttttatggag tctgtatggc ctattcagaggaacagcgtc 49080 ctgaggcgca actcggtaac cagaatcgta acagcctgaa cattcagcaacccggcgaaa 49140 ctgacagcta tgaagcattt ttctctgatc cgaatcgctg gaaggataacagtacgtcgt 49200 tcagcctggg cgatgtattg ccaacaatgg gtaaaggttt cgcccagtccgtccggggaa 49260 caggggaaat ggcccgtgga ctcggtgatg cgatgattca gagcccggtaaaaacagggg 49320 cgcgtatttt aaatgagttc agccgtatgg ggctgccggg tgtcgcaactgtgcaggata 49380 tttttgccgg tggcagcagg ggggctgatg aggtcatcga taccctgcctgatggcaaaa 49440 acgcggttac tgatattgtc ggtaaaggtc tgaaggcaac cggtaaggctgtcagtgatg 49500 gtgccaaagc cactgatgaa tggctgaccg gtaagatgtc gccgggtgcagttcgtgcgc 49560 tgaatacgcc gatgaccgaa ggctataatg attctgcggt ctgggtggcgaagggtgtaa 49620 acctgattgg tgcgcttgta cctgatatgg ttgctggcgg tgtggctagaaaggtgggtg 49680 atgtcacact gcgaaaaatg ctgaccgccg ggctggagaa aaaatacatcgcggcaggga 49740 tgcagccgga aagagccacg gcactggcag cagaagctgt cgataaaaaaatgccggatt 49800 tattccaggc gggcctgatc acccattcca ctgtaagtgc acaggggcagagtgcaatgg 49860 cggcagcaga tgctgttctt aatgctgatt actctgagct ggcgcagtcaccgaaatttc 49920 agcagacgtt tttgtccatt gacgccgacc cgcagcacgc acagcttactgatcgccaga 49980 aaatggatct ggcaaaagag cgtgttgccg atgaggtgcg cgcgcagctggcaaccgatc 50040 ctgaattgct ggctgtgaat gccatggcgg caaaactggg tgacgcacaactgtttaatc 50100 tggtgacacg aggcacagcg aagaccgtta aaagcggcat tgtcagaaatgccacggaac 50160 agggggcgat taatgcggcg cagggcggct attcacgcta tcaggaaaacacggcattgc 50220 gtgagaccgc cggaatgggt gtgtcaccgt gggagggcgt ggctgacgcaacgatcgaag 50280 gtgcagcctt tggtgctgcg atgggggctc cattcggtgc ggttgccggatatcgtggca 50340 gacgtcaggc cgcagaagaa accgccatgc gtgatgctga aaccgtgcagcaggacgacg 50400 cagccccgca accagaatct gttgatccgg tggcgcagca gcgtgaatccatgcagggca 50460 tgaatcgcga gcagcttctg gagcagtatg ctgatgcgga tatggcaacagagggtgacg 50520 catccgcagc tcatcgccgg gaagctgcca gccagttgtt gaatgaactggacgaacaga 50580 cgaagcgaca ggctgtgatg aatgagctga aggcgaagcc gcgttctgaactgcttgagg 50640 aataccgcag actcagccag aaagaggggc gcaccgagac tgaagaacaacagtttcagg 50700 caatacgaga agtcattcgc ccacaacagg aagtgacgcc ggaagcacagtcacagcctg 50760 aaaatgcgga ggatggtaac gggagcattt acccgacggt gcggttccgggacccgaatg 50820 aagtccgcat tgaaattaac gggaatggtg cgtccagacc agcggaacgcattgagaagg 50880 tgcgcccgga caaccgttat ttcacggatg agaaaagcgc catggggagtgatgttttcc 50940 gtaatgccgc cgccaccggc ctgaaaccgt ccgtagtgaa gaaaggcgagaatcagtatg 51000 ccgttgaaat ggataatcct gcgttctctg aagatgtggc aacggaaaccattaacaccc 51060 tggctgacgg agagcgtatt gctgatgctg acccgatgga gcagcccgcgttcatgcgtg 51120 acccgcgatt ccgtggtttc acgggggatg atacggaggt acaggcccgccttgcccgtg 51180 gcaacgcgcc gacggcagag gagcttgtac gttcacagat ggctgaaggtgatgccggtc 51240 cgacagcaca ggagttaact gagcgtccac gcctgcccgc tcccggcgatattcatcccg 51300 gacagggata tccgttaccg ggagaagtgg cgcgtacgcc ggatgaaaatcaggccggac 51360 gtggtggtcg ttttaccaca accggtgagg ttaagggcca gagtttccagaaaggacaag 51420 ctccggcacc ggaaaacgcc gctggtcgcc agggggaaac actcgagggtgacatggttc 51480 gtcgtggtct gccgtcaccg gatgcgcaga acgcgacagc accggtacgtgaagggctac 51540 cggctcctga cattgcgcgt aatgttcgta tgcctcagcc tgaatcacttccccgcactg 51600 tacgggactc actgcctgag cttgcacagc aggcagaagt acgccgacaggccggaggaa 51660 atcgtgacat cccgcagcct gagacaatcg cacctgaatc tgaaacaactgtctctactg 51720 acagggaagc taccgtgcgc ggaggtgaag tcaggggcaa aaaaattgaagactttggtg 51780 aagaaattaa gggcgcggca aaacaccgtt atgcacagct tgctgaaacactgggtaaaa 51840 cgctggaaga cagggattat gccacgcagc cgctgagcaa actgttcccgaaaccggact 51900 acgcaaaact ggcgaacgaa ggtgctgatg ctgataccct ggcaatgatagcgctgtatc 51960 gtagcgatat tccggcgaag acgaaacaca atacggcagg ctggggggagagcataaaaa 52020 aagtacgaca cagtgtatcg gaaatgctga acggaacggt cagcgcgaaacgcctcgcag 52080 aatggatgga aggcagaatg ccctcccgtt acgcggatac ctggcaactgttacgcactc 52140 tgccaccctc acagatggac agggcttctg cttatcgggt ggtatcgggtgtgtatcagg 52200 cggcaggagg gaagcgttac gatccgccac agaaacttta ttcactgcgcaataaggaca 52260 ataaggggag taacctcttt ttctcggaaa gcagggatga attactggcaaaggcgaaag 52320 tctggtttgc agagcaggag gaaaaatcac aggcgaaagg tgatgaaaaaacagcaccgt 52380 caccggatga caaaatccgc tttgacgttt accggaatac ccgcagtggcgatattttta 52440 tcgcttacgg taaaaacaaa atgcgggtga gaggtggctt taagtcagccagtgatgcgc 52500 gtaagtacat tgattcacat cgtgatgagc ttgttcgtca tgtgaaggagatgcgggaga 52560 tttcgcgtga ggagcagcgc aacgccacca accgcgaccg taccggaccagaacgccgca 52620 aggggaatgt ttcaccggag cagttcagtg atgcgtttgg tttccgtggtgtgcagtttg 52680 gtaactacgt ggaaggtccg cgtcgtcagg ctgatttgaa ccgggcttatgactcgctgc 52740 atgaccttgc ggaagtactg aatgtaccga caaaagcgct ttccctgaacggtcgtcttg 52800 gcctggcatt tggtgcccgt ggtaagggta aggcggcggc acactatgagtcaggtgagg 52860 tggcaatcaa cctgacaaaa ggtaacggac cgggtgcgct ggcgcacgaatggttccatt 52920 ctctggataa ttattttggt cgttatgacg tttccaatga cgggaaaattacgtcaggtg 52980 gcgactttat gacggaagca cagcgtgtca ggcgcatatt taaagacggcaggtatgttg 53040 atgctgaata tccggtacgt caggaggttt acgacgcttt taaaggtgtgattcaggcca 53100 ttaaaaacag tgacatgccg cgtcgttcag cgcttctcga taaggtgcgctcaaaaccgt 53160 actggtcaac ggatgttgaa atggcggcac gtgcctttga gcgttatgttcaggataagg 53220 cgcgtatggc tggcgtggag aatgattatc tggtcaatat ccgtaaggcacctgagcaca 53280 acacagataa cacctacgct tatccgacga atgcggaact ggatggcggtattcgtgagg 53340 cattcgatca cctgttccgc accctgaaaa cccgtgagac ggacaagggcgttgcgtttt 53400 attcccgtaa gggcgttacc cgcacacctg aaggtaatct catttcggatgttaaccgta 53460 gtgcggaagc caaaggcagc ccggtcccgc aggttgaagc ggttgcccgtggcgtgatga 53520 gcggcattaa ggacagtgac ctgaaggtcc gtgtggtgaa gtcacagaaagaggctgaag 53580 cgctggcggg tgaattgttc gatggttacg gcagggtgca cgcattctatcgtccggata 53640 aacgagaaat tgtcctggtg gcggataaca tccctgacgg gcggaccgttcgcgagaagc 53700 tgcgtcacga gatcattcac catgccatgg agcatgttgt cacaccagcggaatatcaga 53760 cgattatcaa aaccgtgctg aaaacccgcg acagtgataa cgtcaccatccgtgaagcct 53820 ggcgtaaggt tgatgcttcc tatggtaagg aatcaccgga agtacaggcgggtgaatttc 53880 tggcacatat ggcggagaaa cagccgaata aattcgtggc ggcatgggagcgtgttgttg 53940 ccctggtcaa aggggtactg cgtcgtacgg ggttactgaa gccgacggaactgaacgata 54000 tcagacttgt tcgcgagacc atccgtacgt taggccagcg tgtgcgggaaggttacacgc 54060 cgcgtgagga tggcgcgggc gcatcgtttc agtactcccg tagtggtaaacgtgatccgt 54120 tcaaagtgcc ggaaggtgag ggcgagcgtt atcgtgatga ccttgccagaatgatgaaat 54180 ctctgcgcac cacagattta acggtaaaca tcgggcgtac gccgccggtattgcgtcacc 54240 ttggtgcacc ggatttgccg ctggttattt cccgcgatac tgtgcggaaggccaccaatg 54300 gtgtgaaaca tgtggtgccg atggatgtta tcgagagact accggaactgatgcacgatc 54360 cggatgcaat ttaccgctca gcgacagaaa gaaatgcggt tgtgatgctgcttgatgccg 54420 tggataaaaa tggtgatccg gtggtgtcag cggtacacat gaaggctgtccggtcgcgtc 54480 tggaaatcaa caaggtagct tctgtttacg gtacagaaaa tggaaaaaaactgaagagta 54540 tggaaatgac cgggttaacg ttgtaccgga gagaaaaatt aagccgcgataaccttctgc 54600 acagagggct ccaattgccc aaaggggaac attcttatcg cggctctgcggataaaatac 54660 tctatcctga agatattcgc aaggggccgt attactcccg taccagcagtctgacaccgg 54720 aagagacaat tgcatcgcgt tttgtgcgcc agatgcagga taaattccaggtgctgaaag 54780 ctgttcagga gaatatccgt aaaactggcg gcaaagtgga cgacagtaacaacgcttata 54840 tggcggaaga actcttccac gggaaggcgg aaaacgacct gaacgtgatgaaggagcgct 54900 acgttcagcc actggctaaa ttactggcgg actacaaaat tgcgcaggccgatctggatg 54960 agtacctcta cgcccgtcac gcgccggaac gtaacgcgca tatcgcgaaaatcaacccga 55020 aaatgccgga cggcggttcg gggatgacca acgcggaagc ggcggaaatcatgcagcgtg 55080 tacgtaacag tggcaaacag gcacagtatg accgtctggc agggattattgacgatatgc 55140 tggcccgtcg ccgtgagctt atccgtgagg ccggacttga agagaacggtgtggtggatg 55200 cctggcagaa cgcctaccgt tactacgttc ccctgaaagg tcaggatgttgacggtgtgg 55260 tgtcactgcc ccgtacaggt aagggcttca ccatcggcgg acgtgaaagcaggcaggcca 55320 tggggcgtgc atcccgggca cagtctccgt ccactcaggc gatacaggacctgagcgaat 55380 cgctgatccg ccatcgcaaa aacgaagtgg gtaacgcctt cctgaaactggtgcaggata 55440 atcccgacaa ggattactgg caggtattca ccgatgacag accggataccatgcggacga 55500 ttgcagagcg caaggaccag gaaactggtg aaaccattcg cgaagttgtcgaacgccctg 55560 taccgatggc aatgatggca gaccggtact tcaccaccaa aaagaacggcaaaacgtact 55620 acatcaaact ccatgatccg cgcctgatgc gtgcgatgaa gagtatgggaccggaaacca 55680 gcaatgcctt tgttcgtacg ctggggaaag ttaaccgctt cctggcaacggtgaacacgt 55740 cgtataaccc ggaattcctg gtcagtaact tcatccgtga cgtgcagacggcggtgatga 55800 acctgaaggc ggagcaggga aggagcgacg gtaaactgaa agggctggataacttatccg 55860 ccctggctgt ggtgaaagac agccgttctg ccatgtcagc cgtatacgccagtctgcgtg 55920 gtaaaaccct cacgggaaaa ggtgcacagt ggcagaaggt gtggaaagagtttgttgagg 55980 acggagggaa aaccggctgg tttaacatgg gtgaccttga aggccagcagaaggaaatgg 56040 atcgccttgt ctcactggcg aaggggggat ggaaaggcca gagtatcggtgcatggaatt 56100 cgttccttaa ccttgtcgag gatgccaacg gtgcggttga aaacgctctgcgtctttctg 56160 cctataagca cgcccgtgat gccggtttgt cacgccagca ggcggcgtctcttgccaaaa 56220 acatgacggt gaactttaac cgtcgtggtg agcagggggc gctgatgaattcgctgtaca 56280 tgttcgccaa cgccagcatt cagggaacgg caaatctggt gagaacgctcggacatctta 56340 atggtgaggg acctttactg gagcgccttc gctggaagaa tctgaatgttccgcagaaaa 56400 tcgcgcttgc cgctgtggga gcaggttatc tgcttggctc gcttaaccgcagcgtggcgg 56460 gtgaggatga tgacggggtt aactggtatg acaaggtgcc gtctcatgtgaaagagcgta 56520 acctcgtcat tatgaaatcg gtgttcgggg gcaaggccgg agagtactggagtattcctc 56580 tgccttacgg gtacaacgtt ttcttcctgc tcgggcatac tgctgaaggtgtggcggcgg 56640 gtgacctgac ggcgtcccgt gctgccggta atgttgttgg tggtgtgcttggtgcattca 56700 gcccgattgg cagtgagacg tcggaaacac tgtccggggc attgctgaaaaatgcagcgc 56760 cgaccattct gcgtccgttt gcgaaccttg ccatgaatga aaacttcatgggggcgcaga 56820 tttaccagga gaacatgccg tttggtacac caaaacctga cagccagctgggaagacgtt 56880 caacgccaga agcgtacaag gcgtttgcat cctggctgaa tgcgttctcaggtggcagcc 56940 agtaccgtcc cggcgcggtg gatatcacac cggaatcgct gaaattctggattgactata 57000 tctccggagg gacagggcgc ttcatttcca aaaccacgga tgcggcggtgaaatcgctga 57060 atggtattga tataccggaa cagcaggtgc ccttcctggg gaaaatttcgggggaggtga 57120 tgccgtatgc agaccagcag aagatgtacg accggatgac agagattgcgcagtatcacg 57180 cagagctgaa gagtctgacc ggtgcagaaa gaacggcgtt cattgacgagaacaacggaa 57240 aattgtcgat gaacgggctt atgcaggata cccggaagag actgaaggatttgcgtaaac 57300 agcgtgatgc catttacgcc gacagtactc tcagtctggc gcaacagtcggcgatggtga 57360 aatcggtaga gcgggatatg aaaattgccg tggatcggtt taaccgcgagtacaacaaaa 57420 aagtgggagt ggattaacag aaatggcccc gtacggaagt gcggggctgattaagaaata 57480 aacactcatt gacctgtaat aaccggagct attaacatat agtcagaagagcatttcatg 57540 tgatacagag agccgattta tgtttaatga agaaaaagtt gcgcaaatggcagcgtattt 57600 gctgaaaaag catggcggat ctatgcgttt cattaagctg aagcagcaacggtaatggcg 57660 aatagcattt atgagcaaaa taatcttgat atgttattag gcgatctgatgtaaccggga 57720 tgatgttcac cccttatcgc cggggaacaa taccggcaat cagaattgcggatggaacta 57780 ttcaggccca cgatgatatc gatgaggagt tttttcagcc agtattggatggctttctta 57840 tatccaaata tacgccattt gacatcctcc acgccctgaa ggacggggttttacagcgca 57900 ccggataaga ttcggtgttt tgttgaaaaa tactgtgata acaaacagaaaacccgtcag 57960 taagacgggc ttagcaagct gggacggtta ctttaataat ttcagtgcctttacatccac 58020 ttcaacactg ctcaggtctt tatcaatttc accctcaatt cttactttgtctttcggaga 58080 aacattctga ccggcccata tgctgtcatc gatatccgtg acaattgtcccgctattgtc 58140 acgaaactca taacgttcat cacccacttt tttaacgatg ctcccttcaaggataaccca 58200 tgcatcatcc ttcagttctt ttgcctgcgc tactgttgaa cgctctgcttcaggcccttg 58260 gaaaccgccc tgctgtgcaa aagcgccaaa agacacacca gaaataagtgctgcaatcaa 58320 tacctttttc attcatagtc ctctttcaga gatgaacatt caaacagcattttcagtatg 58380 gtaaagcgcg ggtgcgttga ggatgcctga cacatcagag gtggcgggagattactcccc 58440 cgcttggtct cttacttctc agattcgtag tctacgaaga cagcgacctccgtctgaccg 58500 gttcggattc gcacctcgca gaggtctttc ctcgttacca gtgccgtcactatgacggtt 58560 aaacagatga cgatcagggc gattaacatc gccttttgct gcttcatagcctgcttctcc 58620 ttgcctttcg gcacgtaaga ggctaaccta catgtgttca gcatggattgagcctcagat 58680 taatgttaag cgtcttgccg gacgcgtaat gttaactggg gcttttctctatctgccgtt 58740 ggtgttcatg cccgaggcag atagcctcaa gcacccgcag caattctacttaactctcct 58800 tttcccgcaa accgttttta tctccagcga caaatcgaat acacaaccagcaccaccgcc 58860 attactgttc ctacatttgc gaatgcttca ggccaggtca ttgattcaccttctgctcaa 58920 tatttttaag gtcattttcc gcatacagta ttgcagttct tgctgcccgcaaccgtgctt 58980 tggtgttctt ttcttcacgc tcgaggctgg caacagattc tcggagctgatcgtgtctgt 59040 tatgaagctg tctaatctct ctcaccacag cctcaccatc gttcgcacaccgcagaacat 59100 actgaaaagg gtccacaaca catccgcact gcaagcacag gataacgtgatcctgttcat 59160 gaacctcaat ggcccgatgc ttacagctct gttgtgtgta gttttttctgccagttactg 59220 taatgttcag cagcttctct tcatcgcgct ttggctgcac cagtgtgatgatattatcgc 59280 cttcattttc catcagttca cctcctgcgg cggttctggt agcggcatccagtgtgacgg 59340 tttccacgac gcaccaggaa ttatccaccc atcattagcg tcaggatgccccgggatgta 59400 agtcgcccac ttcattcgcc agtcaccttt cctgtcaaac tccacggcaacaagaacggc 59460 tgttttggta tccggcattc gctcactaca acttatccaa ccatccggagttaccggaga 59520 gttgccagac agcgcattct gcaaccgttc cagcttaacg tattcctgaaccctgtttcc 59580 gtcgcacgcc tgaagccatt gctcagcctt ttgcgcatca gtgtgaaaggcacaagtgcg 59640 accgtcatca aattgcattt cgtagaggtt agcaatctgt tcaaactgcgtgtgtggcaa 59700 cttgtaagtt tggcttacga gttcggcttc cagttctgct atgcgcttctctgatgcttc 59760 aagtaacgcc tgcttatcgc gtagcgcttc ttccagttca gcaacatggcattcactatc 59820 aataaggttg ttctgtgctg ctcccagctc cactctcagc ttcccaaccgtaagcgcaat 59880 ctcctcgttc tcctggtcgc ggcgtttgat gtattgctgg tttctttcctgttcatccag 59940 cagtgccagc acggtagccg ggttagcctc tgctatgaat tcagcgtttgcataagcctg 60000 atcatctgat tcaatcaggc agttaacatg acattccgca atcacgccaccgggttctcc 60060 tttccatttt tggcaaacaa aaactcctgt taaattgccg tgctggttaacagatgtatg 60120 ccctacgatg tagcttcctt tagttgcttt ctctgccttt tcacgcagtgcctgataatt 60180 aatttcgctc acttcgaacc tctctgttta ctgataagct ccagatcctcctggcaactt 60240 gcacaagtcc gacaaccctg aacggccagg cgtcttcgct catctatgggatcgccacac 60300 tcgcaacaat gagtagcaga tggggcatta ctatcggatt tgtatttttgcagggagaga 60360 ttgcgctgca attcttcgat ttcagcggcg ttgtcgatga tatctgccattttcctttcc 60420 ttcaggcatg aaaaaaggag ccgaagctcc tttggtttta gaattcgaattgccttgccc 60480 gcaggctttt cagcattggt ctggcccgct gaactacgaa actcgactggtcaagccgtg 60540 ccgcctctct cagtagcaca tctctgttct tcgtcaccat gtagatggtctcaaacgcaa 60600 tgtcatacag cttgttcgtg tatgaggagt tcagctcttt catgatcggatacaggtgtt 60660 tgctgaggtc ctgggctttt tccatccaga gttgcatgta gcagaggaggatgatttcct 60720 cgtctgtgaa ttgttgctgt ggttctgctt gtgccgactg catgttgcgaagtttctttt 60780 cgcactcgat gaagtatctg cgtatctgtc ggcctttttc gttacgctctaccatcgccg 60840 tttctttggc tgtatcgagg gtaaggtggt agtctttttt ccctcgcccataacctattt 60900 cccgtttctg ggaaatagct atatagtcct gattttcaac gaatccgtactcttcaatac 60960 gttctgtaat ccacgatgca aagcgtttac ctactccaag aaaagtatgtaaatcacggg 61020 cattaacgag aagagtggtt tcgttggcga tagtgccgtt gaatacggggatgcgttgac 61080 tggtcatgat gacctcctta tttgtttagt ttataaccgc cagttagtagctggcggtcg 61140 ggtgtcaact gagccaaata agaagctctg ggcatattcc ccttgcgggtgttgtattac 61200 gcctctccac ccgacctttg tacggatgta actatgccaa attgcaggcataaaaaagcc 61260 gcaaagctat cgggtgcgga gaccgcttat ttgttcagtg cggtcagtatgcgatagctc 61320 tggaggattt gtcaatcagc ggaatattga tgaacgggta acggtaaatgcctcatccag 61380 aaaattaacg caaattcgca tcgcaaactt gcgttaatta tctgaaatatcaagttaaat 61440 ccccttaaat gtttggggca ctattggggc aaaatgtggt tgtttggggcactattgggg 61500 caaaatgtgg ttgtttgggg catatttggg gcaaaaaaga acgtataaagaacggtgtaa 61560 aattgatttc ttcatggtca aaatgaggtg taacagattg aaaaatatatgctcttggac 61620 gatcttcggt aattctgtgt ttttaatgct tcatggtttc aa 61662<210> SEQ ID NO 2 <211> LENGTH: 72480 <212> TYPE: DNA <213> ORGANISM:Escherichia coli <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: 22519, 22524, 22687, 40171, 48548, 49106, 49254, 49258, 49274,49283, 49308, 49318, 49823, 50165 <223> OTHER INFORMATION: n = A,T,C orG <400> SEQUENCE: 2 cagtgtggta catggatatc gataccacgg ctaagtacaaatctggtgtt actaccgtaa 60 aagactcggt acgactggat ccgtgggtgt ttatgttctccgcaggatat cgtttttaat 120 tttctctgca aaacctctgc aaaacccctc tgcaaaactggtcatcaaat gaccagtttt 180 ttccattcct taccgcgtgc gtcgttgtaa atatcggtcattttttgatt cgaatggcct 240 agcaaaattt tggtatcaac cccctgctct ctgaacaatcgctctgataa agatctctgc 300 tcatggaaag agggaggggt gccattagca cgccagttgtaatccacaga atcccgggct 360 tttttaaatg caacggttaa tgttgctggc ttaaccatcccgccgcgctt agctgtccct 420 ttcgcgtgat ggtggtgcaa tagccacgga ctaagaacgcaatcgcggca ggatgacacc 480 acatcatcca gggtgagatt taatttatcg caacgcagagccagagggat ggcaatccgg 540 gttcctgttt tttgctgttc gacatgaaga taaccatcccggatatccga aaattgcatt 600 ttgcaaatat ctgaaaggcg ctggcctgtc atcagtgccagcagcatacc gcgctgtaaa 660 aagtaaccat ccttttccgc tgcgttataa atcatcatccactcatcaaa agtcagtcgc 720 tgtcttgata tccgcacctg cggttttttt gccgattctgcagggttaaa gcctggcggg 780 acatcgcccg tttgctgagc ttcccggaaa acatcgatcagtactttcct gaaaatttgt 840 cccattctgt tatgtcctct ggccttgtac tcttccagtactgataccac atcttttacg 900 gttatggcat ctaacggtct ggtgccaaaa cgttcatcaaataccctgag aggggccgct 960 ttctgtttca gcgtgttgag tttgatctcg ccgttttcatatctttcctg ttgaattttt 1020 ctgtaattat tcagaaaaat ggtaacggtt gatgaaccgccggtatcact aataattttc 1080 tcctgcagac tgagcatttg ttccatttgc tgccgggcaagacggctgtt cgcttctgct 1140 gcaatagttt ctgccagttt ctggtcaata ctgccgagaccgtgattttt gcctgttatg 1200 ggatgcctgt aacgccagta aactttgtta tttcttttgtcaaaatacgg agataatccc 1260 ggaacatcgg ttttatattt tcgcgggcgc gccatcttccagtatcctct tcaaagcagg 1320 gtgatctgtg gcgatcacct ccggcttgtt taccattccgacaaagcgag cttgcggatc 1380 cactcgccag cgtcttccaa cttttttggg gagaggaaatatcattccgg ctttagcgta 1440 tttacttaac gtactcggag ttgggaccgg ttcactgaattcctcttttg cccactcagt 1500 gagcagaata agtcttgcca tgagcgtcgt tcgctaatcatggtcgccgc cactatagct 1560 ggtgggcaac gaccggggtt gaacattaaa aatcagcctgattcgggatc agtttttgcc 1620 agataactga aacgtatttt gcctggtaac gggcgtcatcaagtgcatta tggcgctcac 1680 cttcgaatgg aatagccgtt ctggcatcga agtctatggctttccccagc tcaacgattg 1740 tgcgtacatc gcgatcgttg tagtaacgcc acgggcaggggatcccctgc cgttcgtatg 1800 aacggcgcaa aatcgtgttg tcgaagttgg ctccatttccccagacctga acaaaaaatt 1860 caccggagtt ttcgtcgata aattcccgca attgtaatagtgcatcatct aacgggattt 1920 catcggtcat aatggcagat tgcgcttcgc gtgattgcttaagccaccat ttaatggtgt 1980 cccgatcaat gaccccgcca gcagtttcca gatcgatagtcttactaaat tccggtccca 2040 tatctccggt ttgcggatcg aaaaatattg cacctattgagatgatcggg gcatcaggat 2100 tttttcccat ggtttcaagg tcgatcatta gatggtcacacgtcctgctg gtggatgtga 2160 tttcttgatg accgttcacc ttaattgagt gatctgccgtctcgccagtt tcattatcgc 2220 tatcgtgatg ctgattgccg tcagtgttct ccttgtgtggatgttcagcg ccttccattt 2280 cctccggatc atcttcctga acttcaacct gatactcttcatcgaatgtt tcctggtatg 2340 ttgcgtcgcc catcaccgcg ccacaatcag ggcagttgccgccgccggtc tgaccgcagg 2400 cggtgcagac tttttccggt tcctgttgcg ctactggctcaggttgtttc gtttctggct 2460 cgttttgttg cgtatttggg ctgttttgtt ccgctttctggtcgttctgt tccgtttctt 2520 gctggttctg gttcacagaa tcgcgggttt caatccccttcacccatttc ggatcattcg 2580 ggtcgctaat ccctgcaaca aattcaccac gtgatgcggcgagcaactga ttggcgtcag 2640 gctggctgat attggctgcc tgcataattt tctttacttcgtcagcggta acttttaccg 2700 gttctggttg ttcagaattt tgtgcggtat ttgcattttgcggtaagcct gtgtatgtgc 2760 cattttttcg ggcaaaatat tcttcttttg tgatttcagtagccccggca gccagcgcct 2820 tatccagacc agaaagtttg tttgcgcgac cgtatttttcgccatccttg tcggtgaaga 2880 ggaagtagaa cggcccctca cgctctacag atggttcgacttccactttg cattcggttt 2940 tttcgttgtc cggaattgcc gtttccactg catcagtttctggtactggc gacgagagag 3000 tgtcagttgc gctctgattt gttccttcat cttcaaacacgccctttgta gtcaggtatt 3060 cagtaatgta tttgttcagt gccacagggt ctttgtgaatgtcgatcgga cgttcacgga 3120 caaggccaaa aatagtctgg cggtcgtagc gaagggcatcaggctgtttg cgcattgatg 3180 ccgagatacg cttccagtct tcgcggtcgt tgtcgataacttcatttttt gcccagcgat 3240 ggatgctgcc gtcaatgttt ccggcatcca catcaccaggccagagagcg taggccagtt 3300 cgtcatccag tgttttccat gtctgcttat attcgcgacgaatgactgca gtgacagggg 3360 ggattttttc tgctgagttt tcagtgtgct gtcggtggactctggcgcgg gcgagatcaa 3420 caacagacgt gtattttccg gtttccttgc gttcaccttcgcgacgtttt ttccagatgc 3480 gcatttctgc ctgaatttcg ggccatttgg caccaggcttacatttatgc ttaacccacc 3540 cgatggcatg cagcttaagc tccggataca tggcgttaacttctggcatt ttcatcaacg 3600 cttcaacgat atgtccgtcg aatgttgcca tgtcttcctgcaacaattcc tgcgcgctaa 3660 tcaccatatc aacggtgatg ttttcacatg tgtcgaacttaaccatgaca gcgttctgta 3720 cttcaggggc cagcttgtca aaagcgacgt tcatcggatcggattcagtc tcaaccggga 3780 caaaggaagc agccccctca tcccagcggt tttcctgcatatattcagca tcccaggaat 3840 cgagggcagg gcggggtata ccgggtttat cctcgcaaacaagaaattta taagcgcagt 3900 cctgagcagc cggataatgc tccaggaatt gccagtgaaattttgcgcgg gcgcgacgtt 3960 catcaccggc ttcaatggca gtggctacag cgactgcaccttcttccttt attgcctgtt 4020 cgtccggaat ggcggcgcaa ataaagactt tactcattttgttttacctc attacagatt 4080 taagggtgaa caaatccctg ccattgctgg catataagaatgaaatcgga tgtttattac 4140 ggaactgttt taaagacctg ccgggatttc gttattatcctggtgaataa ctttatcgac 4200 agggtaacag ttaccgggaa ttttctgttc ggttgctgcagtcacacact cctgcattgt 4260 cctgtgaaca ctgactgcaa tatcaactgg ctctccggaaacaagaaaaa ctgtcagaac 4320 aagtgcaaat gctgtattca ttgccagcat cctttttgtatcggacgtaa acgggccagc 4380 attgaaagaa tgcatatttt atttaataac tcccgttcgtgttttctctt gttaatggca 4440 tcttcagtaa atacagggtt actgatagtg acaccaatttcaaaacaacc ttcagacgta 4500 ttaacgtttg gtaataacgt ttccattatc gcgtcctcaacaatgaattt tgtgatgcgg 4560 tgcctggtgc ctccaggtga cgttaaccag ttaacaattaacgccggata cagagaatcc 4620 ccccataaca ctgtttttgg ttttaactgt tccgcgtgcgcttagccgca ttcactgcat 4680 cacaaaattc actttaaaaa gggcggacat cagttcatgggcaaacagat gccgccaaac 4740 gtcaccagaa aattgataac agagggcgtt gcagcggggttgtcacttaa gcgtatggtc 4800 aacctgacaa cccggtgtcc tcaacgggga aggaataaccccgccatact taccgctgcg 4860 ccatttcgcg ttatgctctg acttttcaga gaaatatcctttcagtaaac tgtcagtgcc 4920 ggatgttcac ccgtgtccgg cgcacgcact ccacttcacccgtggagaac tccttaatta 4980 ccaaccctca ggagggtgaa tgttaaaatc aactcttattgctaaatgcc tttatcaaaa 5040 tcgcatggta agcagcattt caataggcga gtctgcagttaaaagtattt tcgaagagta 5100 ctttcccggg catgatttta ataaatggaa taccaaattaccgccagcag tttcaacgcg 5160 tattctgaaa gcaaccgaaa gagcaagtac aattcgcgttaactatttca ttaaagattt 5220 gtgggatctt tgatatccac agagcctaaa gtatgtgcatatggatgtgc tattatgcgc 5280 cctcgcagat ttgcatcatt ttctaaattc actgaacgaaacagggcatc aacaaggctc 5340 tgtacaatgc aaaggcaatc gaagactgtc gccgtttctgttttgattga tgaaagaaca 5400 tggccattca cgcaaacaga aattacccgt ttattaacatcgctttcctg cttttgatta 5460 tcagaaccat atagcccaga aaaagcattg cgcacattacgaaccatatt atcgatggtt 5520 tctttttctg cggtactaag gtcaagagta gccagttgtgaacgaactat attcgatgcc 5580 atttcctgta atggcgttgg taaatcttta aattccattattagcctcgt tggttagcta 5640 ttaacgtggg tatgtaatca ttctggcaat gcttaatgccgctgcttttt ccagattggt 5700 gatatcctgc tccagagcgg acagattttc agcctgcttagccctggctt cattggccca 5760 tttcaggtcc tgcaccgcct taattttctg gtgcatccactcataaagtt catcatcggt 5820 atagtctggc gcgatgatga cggggtctcg tttctgcatgtcggctcctt gtggttagcg 5880 ttgcctgctt tttaaccacg tcaggcgagg tggtatcctctgaggggtct gttactcgag 5940 aggaaattgg ttatgaatac aatcaagttt tcttgcccagaatgtggtgg ctaagtcttt 6000 gacacatcct ttaagccgca gggctctgac agtttcgcgggagccatctg caaaaattgt 6060 ggtcaccttg taactgaaga tgagtcctcg cagttcgatgacgaaatcgt tgacaatatc 6120 ttcggtgcac tcaccagaga ctttctgaag taaaggcgcataccgcttag ttaccgctct 6180 gataactctt acctgtccgg caatggcgct gatatcaatataaagcgcca tcgctgtttc 6240 tttgctgatc cctggacgcc ttccattctg atgtttgactcgcccactga gaaatcctct 6300 gcttcccctt aacgccgggt agcggaactg tttgctgagaacaccgtgcg gtgtcttgat 6360 gagtagaatt tagaatagcc taagagttat ggtcaagctttttgtgtaga aaaacctaag 6420 cttcttgatg taaaaaacac aagtatttga aagtttgtgctttttattac agagagttgc 6480 gaaaaaaagg ggggttattt atttgcgctt cttttgcgagctttgagtag ttcttcaaaa 6540 agtttgttga aattctcaac tcgagcacgc atctctgacaacagagcctt ttgctctgac 6600 tcaggcagtg cgtcgaacag ttgaagcaac tctttttgatcttctgtcag attgactggc 6660 tgattatctg ggatcggttc gcctggttgc ttatcttcatctccaaaaag aagccaagtc 6720 ggcgagcact gaagcgcctg gctcagtgcg aataatctcttccccgctgg ctgtgtttca 6780 tctctttccc attgagaaat tgttacgtga gcgactttgaccagcttacc taatgcggcc 6840 tgagacagtt ttaatttttt tcgcctgtat aagaggcgagcaccgaaggt ttcgtttttc 6900 atattaggta attctaattt ttcttgactt aggtttctctacgatctagt ttccttagga 6960 aaatctaagg gtttcgatat gttgaaaatt gatgctatagcgttttttgg cagcaaaaca 7020 aagcttgcca atgtcgcagg agttaggctg gcaagcgttgctgcatgggg ggaactggtt 7080 cctgaaggtc gcgcgatgcg cctgcaagag gcatccggcggggaacttca gtacgacccc 7140 aaagtttatg acgaatatcg taaggcaaag cgggcggggcggttgaacaa tgaaaatcac 7200 cactgaacag gtttgtgagg ctctggatac ctgggtatgccgaccaggaa tgacacagga 7260 gcaggcgacg atattaatca cggaagcatt ctgggctctgaaagaacgcc cgaacatcga 7320 tgttcaacgc gtcacgttta atgatggcga ggttgatcaacgggcgctgg gcgttaaccg 7380 ggtgaagata ttcgaacgct ggaaagctat cgacaccagagataagcgtg acaaattcac 7440 ggcgctgatt ccggcaatta tggaggctat ccggatcagcgatttcagat tgtattgtga 7500 aattactgac ggaaaaagca ttacgtacat gatcgccgggttaaacaaag aatatggcga 7560 tgtggtggag tccgggctgc tttttgcgga tccagttgttgtggaacgtg agactgacga 7620 gcttatagaa aaagctattg ctttcaagca cgcgtatcgtcagcaatacc aatattactt 7680 tgcagataaa caaatgtctg ccaggggggc gtatgagtatcgatgcacta cgatgggcta 7740 aaaaggtgaa aaccggcagt tcatccagta agtcagtattgacctggctt gctgatatgt 7800 gcggtgccga tttgtgtgca tacccgtctg tatctgcactggcagaagta acggaactga 7860 acaaaaagac tgtgcaggac agcttacgac acctgatggagattgggtta attgttgata 7920 ccggtgagag aaaaggcaga acaaagcaaa ttgtggtgtaccgacttatc ggtgtagaag 7980 aaagtgttgc cgagcctgaa tacacccaaa aacgggagtctttaaaggtg ggtaaaattg 8040 gtgctgttaa taaaaacagt accgaaaatg gttatgtttcagcacaaaac agacccaaaa 8100 acagaactct tagctgcatg gaaaataacc aaagacacccaaattttcca tcaaagacac 8160 ccaaaaacgg atcacggaac ccaaaggaac ccaaagatctaaaccccaca cataacgcac 8220 gcgagagtgc tccgaccagt gagcaggaag ttttgtcgttacaggcagcc ccccctgtat 8280 tcctgtatgg cctgagcgaa cccatcggaa aattcccgatgagcgatagc tggtatccgt 8340 cacgggattt tcgacgacgg gctgcgttgt gggggatggctttgccggag acagaattta 8400 cacctgctga acttgccgcc ttccgggact actgggcagcggaggggaaa gtgtttacgc 8460 agattcagtg ggagcagaaa ttcgcccgtc acgtaaatcacgtcagggcg caggttaaac 8520 cagtcagcaa aggggtaaac catgcagcag caccaggtggcaccgcatca cgggcagttc 8580 aggaaattcg ggcagcacgt gagcagtggg aacgtgaaaacggatttatc agcgacggaa 8640 acggcctgga agctgtggga actcatgggg gtggtttattcgaaccgctg gatccagaag 8700 aacggggccg caccttcgaa gctctggatt gcacagattggcgcgatgac tgagcagcaa 8760 atccggcagg tctgccgcca gtgcatggac cgctgccgggcgggtgaaac atggcctccg 8820 gacctggctg agtttgtggc actgatttcg aaaagcggagccaatccatt cggtctgacg 8880 gtggatgctg tgatggagga gtaccgccgc tggcgcaatgagtcctggcg atacgacgga 8940 agtgataagt acccgtggtc tcagcctgtg ctgtatcacatttgcctcga gatgcgttca 9000 aaggggattg agcgccagat gaccgaaggg gaattaaaacggcttgcaga acggcaactg 9060 acgaaatggg caaagcatgt tagtaacggc ctgagcgttccgccagtccg gcgacaactg 9120 gcggccccca aacgcccgtc ggggccaacg ccaattgagttgctgaaaca ggaatatgaa 9180 cgccggaaag cggctggttt tgtttgagtt gagaagtgattttttaccgg gaggaaattt 9240 atggagactg tttttgacgc actgaaagcg atgggaaaagccacgtcggt atagctggct 9300 gcgcgacttg atatcagtcg tgaagaagta ctgaacgagctgtgggaact gaaaaaggct 9360 ggcttcgttg ataaaagcgt atacacctgg cgtgtggctgataacaacgt tcagcaggaa 9420 cagccagcgc cagaagaaac caccacggca acagaagcgaaaatctcaga gtgcgattta 9480 accgcgacga ttgaacaacg cggaccacaa acggcggatgaactggctac gctgttcggt 9540 acaacatccc gcaaagttgc ttcaacgctg gcaatggcaatcagcaaagg tcgtctgatt 9600 cgcgtaaacc agaacggtaa aattcgttac tgcataccaggtgataattt accagcagag 9660 ccgaaagttg aatcggtagc ggaaaccgat ggtaaagcctttcctcagcc agccggtgtt 9720 gcgttaccgg tacagaaaga tgcaacacag gaagatattaaaacagaaac tgtggcggac 9780 attgtgcagt cgctgccatc gtttactgca acgcgagaagatgatttgat tttgccatcg 9840 ctgcatatgg caaatcgcga actgcgtagg gcgaagaatcatgtccagaa gtgggagcga 9900 gtctgcgccg cgctgcggga gctgaacaag caccgggatatggttgccgg gatttgtcgg 9960 aagtccgggc aatgagcgga tggtgcaggc ctgaaatcatgatactaaca atgaaggtaa 10020 aatgcatcgg cagtctgatt ggtcgtagtg aggcggcggtcaggatgaaa gcccaggtta 10080 agggaataag cctgattctg cggggtgatt ttcaccagtcaacaaaatat ccgtagcgcg 10140 ataacggtca aaaattatgg cgctgacact tttgtgccactggagatgac tgtacctaag 10200 ttcaggggag aagaacacgt ccggtgggat ggtcgggccakatttaaagg gcaggtcatg 10260 gctccagcct gtacgctggc aatggargct gcctggcgggaaattgatat gggaaccacg 10320 ccactcaggg atttactgcc ggtccagaga ataaattcctgttacggtta caccactgtg 10380 atcttgcaag tgcaggaaag taggtctaca cggtaacgcgagtgcgtgta acttttgatg 10440 tcattcccgt agaaacaccg gacaaatttt cgctgacaggtcatgcagaa ggtataaatc 10500 tgcagattat ggacaattac ggatatccgg caagagccggaaaaagcatg ccgcctctaa 10560 ttctcagtgg aagatggact tgattatact cattgcattgtcagaaatag ttatccatta 10620 aaggctggct attccaaaca ggatgttgat tacaaaaatgtaatcaacat gtaaggttta 10680 tactcttcaa tatgcgtata attttcctta ttttgttgactttaaataac aagctatgca 10740 cgaggtaaag tcsgataagt ttatctggat gtaatatatattatttgtag tgtttataac 10800 tttatttcat gataaccaat aaaaggagtt ttttatgaggaacataatgg caggtttttt 10860 aatattcctg tcttctgctg cttatgctga tatcaatctgtatggtcctg gtggcccgca 10920 tacagccttg cttgatgcag ccaaacttta tgccgaaaaaacaggtatta tagtgaacgt 10980 tcattacggc ccacagaaca aatggaatga agatgccaaaaaaaatgcag atatcttgtt 11040 tggcgcatca gaacaatctg ctctggctat cattcgggaccataaagaca gcttcagtga 11100 aaaagatatt cagcctcttt atctgcgaaa aagtattttactggtaaaga aaggtaatcc 11160 taaaaatatc cggagtattg acgacctgac cagacctgggattggcgtaa ttgttaatga 11220 tggtggtggt accagtaata catcaggcac tggcgtctgggaagatattg ccggacgtaa 11280 agggaatata gaaactgtcg ccgcaatccg aaaaaatattattttatatg cgcccaatag 11340 cggaactgca cgtaaggctc ttgagaatca gcctggagcagatgtctgga taacctgggc 11400 tgactgggca gccagtaatc cagaaattgg tgatgtcgtggaaatagcgc cagactacgt 11460 gatatggcgt gatatgaaca ttacagtacg tcaggatgcaaatgatgaaa cccgtcgatt 11520 tgcagaatgg ctacaaaccg atgaagcggc gcctgcattcaaaaaatatg gctggaccag 11580 gaaaggcact tgacatcctc gtccttcagg acgtggattctttttccgga tgccgcgcca 11640 gcggcatgta ggggcagctc acaaaacgga aaaaattgtacgctaagcct cgccaggtga 11700 actgaattca ttccgatatg ggaattccca tatcgggcgaaaacggtttg ctgtaacggc 11760 agagttaagt aggattgctg cgggtgcttg aggctatctgcctcgggcat gaacaccaac 11820 ggcagataga taaaagcccc acccgactat aaatcgaagtgaggccccta tatgctcgtc 11880 acatatagat tgcctcttac ggaccgaaag gtcaaggagaagcaggctat gaagcagcaa 11940 aaggcgatgt taatcgccct gatcgtcatc tgtttaatcgtcatagtgac ggcactggta 12000 acgaggaaag acctctgcga ggtacgaatc cgaaccggccagacggaggt cgctgtcttc 12060 acagcttacg aacctgagga gtaagagacc aggcgggggagaaatccctc gccacctctg 12120 atgtgtcagg catcytcaac gcacccgcac ttaacccgcttcggcgggtt tttgttttta 12180 ttttcaacgc gtttgaagtt ccggacggcg ccggaatagaatcaaaaata cttaagtagc 12240 gcgcagggag aagagggatg gaccccgaac aggggagtgctatttatctg gaaggattct 12300 gttgatgaga atcgaaraat tacgtgaaat ttttagtgaagatggcctct atactgtgcg 12360 cgttgagaag ggcgctattg tcagccactg ccgtattaaatgtttacagt ctcaacaaag 12420 gaagagtgga gctgcgttaa ttcattttgt ggatgggcttgtgacggatg gttttatttt 12480 gcgtgcaaat gaatttgtca catcgttgcc gtctctgaaagaagctggga ttaaggctgg 12540 tttttctgct tttgaagatt agtgaattca tctacaattcagcgcagggc tgaacccctg 12600 ttgagtaaca ctgtgccacc ggagaaagcc gatggcgcaaaattccagac tacacaattc 12660 tgataattca gccgtctttg ccagcaggca cgggcggcgttctcatgcat tcaaatctga 12720 ctggttccgg cacgccccat gcactgaaga acaggccgaatggctgattc agaactaccg 12780 cagacgtggg tatgagttta ggaaagccct cagcctcgattatcgtcact ggataatcta 12840 cgtcaggctg ccgtactccg agcgcccacc gcgtccgtcccgcacattcc agcaacgcat 12900 ctggaggtaa cgtgcgggta ttacttcgac ctgttccggtaccggaactt ggggtggtgg 12960 tccttaaacc aggccgtgaa tccattgcag gtattccataaccctcgagt gcttgtggag 13020 ccggagccga aaagcatgcg cggtctgccg tccggcatcgtccctgccgt tcgccagccg 13080 ctggcggagg ataaatcatt actgccgttt ttcagcaatgagcgtgtgat tcgtgctgtt 13140 ggtggcgctg gtgcactgtc tgactggctg ttgcgtcatgtcaaatcctg ccagtggcct 13200 catggtgact atcatcacag tgaaaccgtt attcaccgttatggtaccgg cgcaatggtg 13260 ttgtgctggc actgcgacaa ccagctgcgt gaccagacatccgaatcact cgagcaactt 13320 gctcatcaaa acctgtcagc atggatgatt gacgtcatcggtcacgcaat aagcggtacg 13380 caggagcgtg aattatctct ggctgaatta tcctggtgggcggtccgcaa tcaggtggcg 13440 gacgcgctac cggaagcggt attacgtcgt tcgctggggttgcgtgcgga aaaaatccgc 13500 tcaatgtacc gtgaaagcga catcgtaccg ggagagcagaccgccaccat catactgaag 13560 cagcgcacaa aaaatcttgc gccgctgcct cacgcccaccagcaaaaccc accacaggaa 13620 aagacggtgg tcagcattgc cgttgatccg gagtctccggaatccttcat gaaacgacct 13680 aaacgtcgcc gctgggtaaa tgagaaatac acacgctgggtaaagacaca gccgtgtgcg 13740 tgttgtggta agccagcgga cgatcctcat catctgattggtcatggtca gggcggaatg 13800 ggaacaaaat cccacgatat tttcacgcta ccgctgtgtcgggagcatca caacgagctt 13860 catgcggatc cgctggcgtt cgaagaaaag catggttcccaggttgattt aatttttcgt 13920 tttcttgatc acgcctttgc gactggcgtg ctcgggtaaaagaggtgact gatgctcata 13980 gatttggttt taccttaccc gccgacggtg aacacttactggcgacgccg tggcagcaca 14040 tattttgtat caaaagccgg ggagcgttat cgccgggcagtggtgcttat tgttcgccag 14100 cagcgactga aattaagcct gtccggacgg ctggcaataaaaattattgc agagccaccg 14160 gataagcgtc gtcgtgacct ggacaatatt ctgaaagcaccgctggatgc gctgacgcat 14220 gcgggagtgt taatggacga tgagcagttt gatgaaatcaatattgtacg tggtcagcca 14280 gtatctggtg gacggctggg tgtgaagatt tacaaaattgagagtgagtg agcgtaaata 14340 tgatatatcc ggaaattaca ggcaaaagcg gcgaacatttacgcctgaac acgctggaag 14400 cagtctggat ccaggggaaa ttacggatgt gggggcggtggtcgtatatc ggtgggggta 14460 aatccggaaa tatgtttaac cggttactgg tttcgaaaaagctgacgaaa acagcagtta 14520 atgaggtttt acgcagcatg aagaaatccg ggctggaaaaaccggaactt gaggcatttt 14580 ttcgggatat gaccagaggg aagcagaaga gctggttgtcacattgtaca gacacagagg 14640 cgttgattat tgatcgcgtt atcagtgagg tgcttggggaatatcccggg ctaatcaata 14700 ttctccggca aaggtacgaa ggacggggaa tgagtaagagaaaaatggca gaatgtttaa 14760 atcgtactca cccggaatgg tgtttcagca catgtgagaaacgtattgca ggttggttag 14820 ccgtggctga acacatgctt tatgtaccta tgcacgattcatttcgataa aaaaagcttg 14880 cttttttacg cagaaacagc ttgaattcct gtaagcttcgcaaagctgta tcgcgaggcg 14940 aaatgcaagt tttttcgcac aaggaagcca ccggaaggtggtttttttgt gtccgtaata 15000 tacagcagcg caataaattc gctggtggtt attaataccgttctttcagg ttgctggctt 15060 tttcgacaag agttattggt gtgtcacgtt aaccggaaaagggaaaaaga catgctgaaa 15120 cagcaggata tgacagaaac cgccagagtg gtgtttaatgaattaagcgt taccgacccg 15180 gcgacagtcg gggagattgc gcagaatact tacctttcacgcgaacgctg ccagttaata 15240 ctgacccagc tggttatggc gggtctggca gactatcagtttggttgtta cagacgcctt 15300 cagtcctgaa ggctttttta tttgtggtaa atgggcggctggtgggtgta aggggcaccc 15360 accagccatc tgctcatgcg ttgggttcac aagcaaacctcaggcccact gctttgcgca 15420 aaagcagaat gagcctatca gagacaggct taatgatccatgcttaatac tgtaaaaata 15480 tccagttgtg agttaatcaa cgccgactgc ctggaatttatgcggtcgtt acccgaaaat 15540 tctgttgacc tgatagtcac ggacccgccg tacttcaaagtgaaacccga gggctgggat 15600 aaccagtggg cgggtgatga agattacctg aagtggctggaccagtgtct tgcgcagttc 15660 tggcgggtgc tgaaacctgc cggaagtctt tacctgttctgtggccatcg tctggcatct 15720 gacaccgaaa tcatgatgcg tgagcggttt aacgtgctgaaccatatcat ctgggcaaag 15780 ccgtccggac gctggaacgg gtgcaacaag gaaagcctgcgggcgtattt ccccgccaca 15840 gagcgcattc tgttcgcaga gcattatcag gggccgtatcgtccgaaaga tgccgggtat 15900 gaggcgaagg gtaggacact gaaacagcat gtgatggccccgctgattgc ttactttcgt 15960 gatgcgcgcg ctgtcctggg gataacggca aaacagattgcagatgccac aggaaagaaa 16020 aacatggtgt cgcactggtt cagtgccggt cagtggcagctgccgaacga aagcgattat 16080 ctgaaattac aggcactgtt tgcccgggtg gcagaagagaagcatcagcg gggtgaactg 16140 gaaaagcccc accaccagct ggtggatacg tatgcctctctgaaccgaca gtatgcggag 16200 ctgcagagtg aatataagca tctgcggcgg tatttcggtgtgacggtgca ggtgccgtac 16260 accgatgtgt ggacgtataa accggtgcag tactatccagggaaacatcc gtgcgaaaaa 16320 ccggcagaaa tgttgcagca gataatcagc gcaagcagtcgtccgggaga cctggttgca 16380 gatttcttca tggggtcggg gtcgacagtg aaagcagcgatggcgctggg acgtcgtgca 16440 actggcgttg aactggagac tgaacgtttt gagcagacggtgcgggaagt acaggattta 16500 atcattcgta acggatgaga ttgcggagtt aatcatgcgtcgttattatt cagcaatcgg 16560 ccctttagct cagcggtgag agcgagcgac tcataatcgccaggtcgctg gttcaaatcc 16620 agcaagggcc accaaccgcc actagctcat caggaaagaacgtcaccctg tgcgagattc 16680 ggagtccccg gtggcggtcc attatcggta ttctgcgttgttagctcagc cggacagagc 16740 aattgccttc taagcaatcg gtcactggtt cgaatccagtacaacgcacc acaccacact 16800 tatctgccct gactctcttt tgcgggcttt ttattacaggaaagacaccg gacagtgaaa 16860 tgttaaatgc ctcacaattc aggcagttga ctgttgcctgacatgctgag cgtttgttaa 16920 aaaaatcctg catgatgaat ccccctgagc ggcggggcataatgacagat gtttggttgc 16980 gtattgtata ggcaagttgc ggattctgtc tggtcattgcagaattcacc gggaggcacc 17040 cggcatcatg ctgtatacag agattaggca tatatccaggcttctcatcg caggagcctt 17100 tttacatgca aaaaaaagcc cgagtgggtt cgggcaacagcatgagatac ttgcattgtc 17160 atttttatcg tgtggatttt aaccagggtt tataaggctgcgcaactgcg cggccttttt 17220 cgttttgcgg gctgcggttc tcctcttttg attctccttgtggccggacc gtggcccgca 17280 actgttgagg aaaatcccgg aaaggggagg aataatgacatttaaacatt atgatgttgt 17340 cagggcggcg tcgccgtcag accttgcgga aaagctgacacacaaactga aagagggctg 17400 gcagccgttt ggtagtcckg tggccataac cccttataccctgatgcagg tgattacagc 17460 agaaggtgat gtggtggtca gtggtgcaac tgagccggattggtactacg tcatcgtact 17520 ggccgggcag tccaatgcca tggcttacgg tgaagggcttccgctgccgg attcatacga 17580 tgctccggat ccgcgcatta aacagctggc gcgccgcagtacagtkacgc cgggtggggc 17640 tgcctgcaga tataacgata ttattccggc cgaccactgcctgcatgatg tgcaggatat 17700 gagtacgctg aatcatccga aggcagacct gagcaaagggcagtacggct gtgtcggcca 17760 gggsttacat attgccaaaa aactgctycc gtatatcccgaataacgcgg ggatcctgct 17820 ggtaccatgc tgtcgtggtg gttcggcatt cacccagggcgcggagggga cattcagtgc 17880 ggacgcgggg gccagccagg attcggcgcg ctggggtgtgggtaaaccgt tatatcagga 17940 cctgattgcg cgcactaaag ctgcattaca gaagaacccgaaaaatgtgt tgctggcggt 18000 gtgctggatg cagggagagt ttgacatgag cgccgccacccactgcacag caacctgcgc 18060 ttgtttacag ccatgctggc acagtttcgt gctgacctctccgtgtttaa cgcgcagtgc 18120 catggtggca gtgctgcaga tgtgccgtgg atttgtggtgacacgacgta ttactggaaa 18180 aatacmtacg syacccagta cracaccrtk tacggsgsgtayaaaaacag ggagagtgag 18240 ggcgtttatt ttgtgccctt catgacagac ggtaacggcgtcaataccgc cactaacgcg 18300 ccggcagaag atccggatat tccggcatca ggatattacggtgcggcatc gagaacgaat 18360 ggaaaccagg tatcatcaaa ccgcccgaca catttcagttcatgggcgcg caggagcatt 18420 attccggatc gtatggcaac cgctattctg aacgcagccgggcgcacctc agccttcatc 18480 agtggtaagg caccggaaat caaaccctcg cccggcggcaacacgccatc gggtccgtct 18540 gcagatacgt ccgttcgcac aatctccctg ctgccggcagccggagaggc tgctgcgcag 18600 ggctggagca ttaaggatgg cggaattcag ttgtcagatggtgtatttaa gatcaccarg 18660 cagarcaata aaacctggtc cctgacgcat ccggtggatgacgcaattac cctgctgaca 18720 cagggcggca gactgacctg taagttccgc ctgtcaggcgcactgaccaa caatcagttc 18780 gggctgggga tttatctgta tacggatgct cccgttcctgatggtgtggc gatgacgggt 18840 accggtaatc cgttcctgat gtcgtacttt actcagaccactgacggcag agtgaatctg 18900 atgcatcaca ggaaagccgg aaacacgaag ctgggggagttcggcgatta cggtaaccga 18960 ctggcagacg ctggagctgg tgttcaccgc cggcagtgccacggttactc cgaaactgaa 19020 tggagtggct ggcccggcat tccaggttat aaaagacagtctgacactgg gactgaatgc 19080 gctgacgctg acggatgtta caaaaaatgc agcgtatggcgttgagatag aaagtctggt 19140 gctggagata aatgcaccgg cagcataata aaaaaagagccagcgactga cctgaaagaa 19200 gacgctggct aaaaggcctt atatgtttgt agagacttatttttcacaga cagcaatgat 19260 gcctgtcaat atattatcaa tatgcggatt gtttcagttacagatgcttt attaaggaaa 19320 aaaacagcca gcactgactt tcggtggaga ggtgctggctcagaaggata gttggatttc 19380 acatgatact tatgcctggc ggtatatttt ctgacagacagtgacgggtg ttgtcaagat 19440 attgtgtcat ttataacctg aatcaggggg tggccggaatgttatctggc atttttagca 19500 gagcctgaat gccataatca cggctcccgg cgttggccgtcagtgggtga cactggcggc 19560 ttttttgttt tyctttactt tcattttctg tcggcggtgacggagacata catcagatgg 19620 aaaaaatcac aacaggtgtg tcatacacca cgtcagcggtggggacggga tactggttac 19680 tgcagctgct ggacaaagtc tctccgtccc agtgggtggcgataggcgtg ctggggagtc 19740 tgctgtttgg gctgctgaca tatctgacga acctgtatttcaagattaaa gaagaccggc 19800 gtaaggcggc gcggggagag taaagtgatg aagaaaaaatacgaactggt tgttaaaggg 19860 ataaataatt acccggataa gattactgtt actgtggcacyggaaattgg tgggtatccg 19920 tcactgttgt tgccagatgt ggcgattagt cttgaccgtactgaaggtgc cacgctggag 19980 ttttacgaag ctgaggcgaa aaagcaggcg aagcagtttttcatggatgt tgctgccggg 20040 ttatgtgaag gggatggtcc gttaccggaa aagcgtcccgtaattttaga ggcgcaggat 20100 gtgttgataa cctacagagg aaaactaccg ggaataattacgggttctct gaagactcca 20160 ccgctggcct gaagacttaa catatccagg gatttgaaatcgataaaccc tgataaatat 20220 ccatgaacgc aaaaatcaga tacggcctgt cggctgccgttctggcgctg attggtgcag 20280 gggcgtctgc gcctgaaatc ctcgaccagt ttctggatgaaaaggaaggt aaccacacca 20340 cagcataccg tgatggtgcg ggtatctgga ccatctgccgtggagccacc cgggtggatg 20400 gtaagcctgt gattcctggc atgaagctgt cgaagggtaagcgtacagcc tgaaccgtct 20460 ggtcagaatc tgacgaatta gacaaagtgg tgtccaccaaataagtagtg ggaaccaaag 20520 tatcagatat gcagaaaaat gtgactcccg gcaggcgaaaaggctgccct aattatcctc 20580 ccgaatttaa acagcagctc gttgctgcct cctgtgaacccgggatatcc atctcaaaac 20640 ttgctcttga aaatggcatt aacgccaatc tgttgttcaaatggcgacaa caatggcgcg 20700 agggaaagct gctattacct tcttcagaga gcccccagctacttcctgtg actctcgatg 20760 cagctgccga acagccagaa tcgctcgcag aggacccggaaaccctcagt atcagctgtg 20820 aggtaacgtt ccggcacggg acgctccgct tcaatggcaatgtcagcgaa aagctcctga 20880 ctctgctgat acaggaactg aagcgatgat cccgttaccttccgggacca aaatttggct 20940 ggttgccggt atcaccgata tgagaaatgg cttcaacggcctggctgcga aagtacaaac 21000 ggcgctgaaa gacgatccca tgtccggcca tgttttcattttccggggcc gcagcggcag 21060 tcaggttaaa ctgctgtggt ccaccggtga cggactgtgcctcctgacca aacggctgga 21120 gcgtgggcgc ttcgcctggc cgtcagcccg tgatggcaaagtgttcctta cgcaggcgca 21180 gctggcgatg ctgctggaag gtatcgactg gcgacagcctaagcggctgc tgacctccct 21240 gaccatgctg taaatctctt tatcctggtt gtcacagaataagcccggta aaatacgggc 21300 ttatgaacga catctcttct gacgacatct tcctgctgaaacagcgcctg gccgaacagg 21360 aagcgctgat ccacgccctg caggaaaagc tgagcaaccgggagcgcgaa atagaccatc 21420 tgcaggcgca gctggataaa ctccgccgga tgaacttcggcagtcgttcc gaaaaagtct 21480 cccgccgtat cgcacaaatg gaagccgatc tgaaccggcttcagaaagag agcgatacgc 21540 tgactggtag ggtgtatgac ccggcagtac agcgtccgttgcgtcagacc cgcacccgta 21600 agccgttccc tgaatcacta ccccgtgacg aaaagcgactgttgcctgcg gcgccgtgct 21660 gcccgaactg cggcggttca ctgagctatc tgggcgaggataccgccgaa ctgaaccgcc 21720 ccggttttcc tggagagtgt tttatctgtg aactcaggctgccagatcat cgtttccgat 21780 ggaagcataa taagcttttt ctgcttctgc cggaggagtatggcccagcc ttcccagcaa 21840 tcgtcgattg ttataccagt ccacccacgt tagtgtggccagttccactt ctgcacggtt 21900 tttccagctc ttacggtgta ttacctccgc tttgtaaagaccattgatgc tctcagccat 21960 cgcgttgtca tacgagtcgc ctgtactccc tgttgatgccagtaatccgg cttcttttag 22020 tcgctccgta taggccagtg acacatactg agagcctttatcgctgtgat ggatggtgcc 22080 agacggacga cgggcccaca acgcctgctc cagcgcatccagcacgaatg tcgtttccat 22140 agacgatgag acccgccacc ccacgatgta tccggcaaacacatcaatga taaacgccac 22200 atagacgaag ccctgccatg tgctgacgta agtaaaatcagccacccaca gctggtcagg 22260 tcgttctgcc acgaactgac ggtttacgcg gtcgcctgcggcaacggctt tccggctgat 22320 ggtcgtacgg acctttttac cccggagaac accggcaagtcccataaccg ccatgagacg 22380 tgccactgta catctggcca ccctgattcc ttcccgtaacaactgacgcc agactttacg 22440 cacaccgtac acctgatgat tttcatcgta tacgcgctgtatttttctct tcagccagtc 22500 gtcgtgctgc gcacgggcnc tgcntttatc cggatgatgtcgctgttgct gacaatggta 22560 atacgttgac ggggcaatat gcagttcgct gcataccggtccgaccccgt actgctcacg 22620 cagcttatcc agcagtggca tcattttttc cagaggcggtcgaactccgc cttcgcaaaa 22680 taagcgnaag cctggcgaag gatatcgtta ctgcggcgcagttcacgatt ttcacgttcc 22740 agctctttca gacgctgacg ttcagcgctg gtgagcccaccatcaccgcc cccggtatcc 22800 cgctcatgct ggcgaaccca gacacgcaga gtctccggcgtacagccaat ctttggggca 22860 atggaacaaa ttgccgccca ctgtgagtca tattcatcctgactttccag aaccatacga 22920 atcgcccgct gacggacttc gggggaaaaa cgagtatttttagtcatcct gtttacctct 22980 ttctcaggga gtttagtctc caggatttcc ggggcggttcacagggtaat cacgcaggtg 23040 gtgatactgg atgcggataa aaagcagata cagtgcgtggtaagaccgct gcaaatcctg 23100 cgtgctgacg ggacgtggga aaatattggc gggatgaartarcccgacag cttcacaaaa 23160 accggagtcc ggctccggtt tttgttgtca tgtatgggggctgtttgtta tgactccctg 23220 tgtttggaat gaatatttaa atagggagtt ttgtcatgccagttaacatc agctattgca 23280 tccaattcat tttccacctg aatgcaggtt cttcgtgctcaaatggccgc cagtggcggt 23340 ggagaaatta cagtaggcgg gcagacggtc cgtatcacatatagtgaaac ggatggtcgc 23400 tttctggcga gtgggggcaa taacagtttg ctttctggattattacttac agggcttaat 23460 ggtggtcctg aagcgctcag ggatataatg ttaagaatggtttcaggttc aggtaacaca 23520 caatcacatg gtgatattga ggggaaaata tcacaatgtaagttttctgt taatacggag 23580 agccttcagt gtccatccga ggcggttcga tgcccaattatactggataa accagaagaa 23640 ggtgtgtttg ttaaaaattc agaaggttct ttggtttgtaccttatttga ttcggtttct 23700 ttttctcatt tggttcgcga cggtgggaag cacccgctaacacgagaacc aataacygtc 23760 atcaatgatt gtaagtcaag aacaatgtat ttatgaccaaaccaaaggaa actttgtcat 23820 aaaggataag tgaaataaat attacccaag ctatatgttaactgccagtt gcttatatga 23880 aatgctacag atgttcaggg tataaggatg tggtaattggtgtactggat gcagctaaaa 23940 agcatataca gtgagtggtg aggccgctgc aaatctggcgtgctgacggg gaaatattgg 24000 cgggatgaaa tagccgacgg attcacaaaa accggagtccggctccggtt tttgttgtca 24060 tgtcagggag atgtttgtta tgaagcccag aggaaatatttatctgtatg aaggaatatg 24120 gtaatgcctg gattagtatc atatatatca tcgacttcattcgcgaatga gatggcggag 24180 atgcgtcagc aggtaatgga agggcagatt ggtggatttctcctgggagg ggagagagtt 24240 agagtttctt atttatttca attgcattaa tatcttgaatggattacata gagttaatgt 24300 atccagtact ccctattctc ttgcatataa tacatgttgcaacttacatc tcagcgctat 24360 gaaaaaacac cacctctctc tttatgaaat tctggatttaccaagcgcta atttatcatt 24420 tcaatctacc tttaagtatt gcatttatct ccctacgaggtcatacttta ggaagttaaa 24480 tatgaatgat aatataccta cagcgcgaaa tcacaaacaatcgacttgta ttacagaaaa 24540 aacatgccta tatttttaaa cttcacagca ggaagtatcttacctgagaa tgagctagca 24600 tctttacgtt atattgtgca gcaaaatcaa aatgatactgtaatcataaa agaacgttat 24660 aaaatggata tccgttatat cgaatcagtc aatggttttacagtaaatcc tgtatgcagt 24720 aatcatttct ccatatttat ggcgagacaa aacactattgctcgcaacct ggaacagcag 24780 atcaacaacg gacgaagttt tgcacaaata tctcaggattttatgcttca attatcttca 24840 aatataggat ggaaaaaagg ggccgaaaac gcccttaaaaataaaatcca ttctcattca 24900 tttgttgtaa atcctgatga attctcttgc gacacacaatttcttaagtg cccaataaca 24960 ttatgcgttc cagaaaaagg ggtttttgtc aagaatgcactgaactccaa catatgcact 25020 ctttatgata agtctgcgtt catgaatctc acaagagaacatctacccca ccctctcagc 25080 agggaaaaga tagtaaaaga aatgattatt gaaaggaatatgtgttattt tgacaccata 25140 agtcagcatt tcataattat ggatgcagac caacaagaaacagcattgta aataaaatgt 25200 aataattaca tactattagt gattctcatg catcgtaagcggctcgccag aaccgtattg 25260 atatttactg aggagcctgt acatagattt gtgtwattgcctgattttga tatgttcaat 25320 ccaacatcaa aagcaggtta atttatggac gaaaaacagttgcaggctct ggctaacgaa 25380 ctggccaaaa atytcaaaac ccctgaagat ctcagccagttcgatcgcct gctgaagaaa 25440 atcagcgttg aggcagctct caacgctgaa atgtcccaccatctgggcta cgataaaaac 25500 cagcctaaac cgggtgccaa ctcccgcaat ggctattccacaaagaccgt tatcaccggt 25560 gatggccacc tggaactacg cactccgcgc gatcgtgatggctcttttga accgsaactt 25620 gtgaagaaaa accagacccg gatcaccggg atggataaccagatcctgtc gttgtacgcc 25680 aaaggcctga caacccgcga aatagcggct gcgtttaaagagctgtatga cgctgatgtc 25740 gtaagcgtac agcgagggcc gtattgacgg ggatgtgttattcagctggc agtgctatgc 25800 gccacggaag cagttcgctg acccggttga ccggccagtcttgctattga cgccaagcac 25860 atggcgaagg tagctttctg gatccacgtc atttcagtttgcacgtcccg atcaggctgt 25920 acagtagcgc tccccgctca ccaccatgat cagagccgaagaacaggaag tttttacgac 25980 ccagactgac cgcccgcagg gcattttcag cgatgttgttgtcgatttcc acccagccat 26040 cgttcgcata gtacgtcagt gccggccact ggttaagtgcgtacgcgaac gccttcgcca 26100 actctgagtg tcgcgacagg gtcttcatct tttcacgcaaccagctttcc agggatttca 26160 acaacggttt cgtttttcgc tgacgttcag caagccgctgctctgccggc attcccctta 26220 tatccgcctc tatggcgtac aactgaccga tctgctccagggcttcttcc gtcagtgctg 26280 acgggatgcg gacgtgcaca tcgtggatct ttcggcgggcatgagcccag caggcagctt 26340 ccwgttatcc caccattgcg atacagctcg ttgaacccggcgtacgcatc cgcttgcagc 26400 acaccgctga agcaggcaag atgagtctgc ggatggatgccttttctgtc cgggctgtaa 26460 gcgaaccaca ctgcaggtgc caacgctgac cctgcattgcggtcatcacg aacatacgcc 26520 cacaaccgcc cggtcttcgt cttcttatta cccggcagcagtacctggac cggggtatca 26580 tcggcatgga gtttgccgtc agtcatgaca tagccatgaagcgcctcttc cagcggagac 26640 agcagccggc agcatgcatc cacccagccc gacagcagtgaacgcctcag ctccacacct 26700 tgccggccgt atatttctga ctggcgatac agcggggtgtgctctgcata cttcgaggtc 26760 agcacgcggg ccagcagccc cggtccggcg ataccccgctcgatgggccg cgaaggtgca 26820 ggtgcctgca cgatggcatc gcactgagta caggcatgtttttcccgtac cgtccggata 26880 acccggaagg ctgctacgca tcaactccag ctgttcggcggtatcctcgc ccagatagct 26940 cagtgaaccg ccgcagttcg ggcagcacgg cgccgcaggcaacagtcgct tttcgtcacg 27000 gggtagtgat tcagggaacg gcttacgggt gcgggtctgacgcaacggac gctgtactgc 27060 cgggtcatac accctaccag tcagcgtatc gctctctttctgaagccggt tcagatcggc 27120 ttccatttgt gcgatacggc gggagacttt ttcggaacgactgccgaagt tcatccggcg 27180 gagtttatcc agctgcgcct gcagatggtc tatttcgcgctcccggttgc tcagcttttc 27240 ctgcagggcg tggatcagcg cttcctgttc ggccaggcgctgtttcagca aggaagatgt 27300 cgtcagaaga gatgtcgttc ataagcccgt attttaccgggcttattctg tgacaaccag 27360 gataaagaga tttacagcat ggtcagggag gtcagcagccgcttaggctg tcgccagtcg 27420 ataccttcca gcagcatcgc cagctgcgcc tgcgtaaggaacactttgcc atcacgggct 27480 gacggccagg cgaagcgccc acgctccagc cgtttggtcaggaggcacag tccgtcaccg 27540 gtggaccaca gcagtttaac ctgactgccg ctgcggccccggaaaatgaa aacatggccg 27600 gacatgggat cgtctttcag cgccgtttgt actttcgcagccaggccgtt gaagccattt 27660 ctcatatcgg tgataccggc aaccagccaa attttggtcccggaaggtaa cgggatcatc 27720 gcttcagttc ctgtatcagc agagtcagga gcttttcgctgacattgcca ttgaagcgga 27780 gcgtcccgtg ccggaacgtt acctcacagc tgatactgagggtttccggg tcctctgcga 27840 rcgattctgg ctgttcggca gctgcatcga ragtcacaggaagtagctgg gggctctctg 27900 aagaaggtaa tagcagcttt ccctcgcgcc attgttgtcgccatttgaac aacagatttg 27960 cgttaatgcc attttcaaga gcaagttttg agatggatatcccgggttca caggaggcag 28020 caacgagctg ctgtttaaat tcgggaggat aattagggcagccttttcgc ctgccgggag 28080 tcacattttt ctgcatatct gatactttgg ttcccactacttatttggtg gacaccactt 28140 tgtctaattc gtcagattct gaccagacgg ttcaggctgtacgcttacct gatgtctcac 28200 cggcgctggt atcgaaggtc accgatgccg ttatggagcaggtagtcgaa tggcaaaacc 28260 ggccactgga tgcggtctat cccattgttt atcttgactgtattgtcctg aaggttcggc 28320 aggacagtcg cgtaatcaat aaatccgtat tcctcgcactgggtatcaat atcgaaggcc 28380 agaaagagtt actgggtatg tggctggccg aaaacgagggagcgaagttc tggctcaatg 28440 tgctgactga actaaaaaac cgcggcctga acgatattctcatagcctgt gtcgacgggc 28500 tgaaaggctt cccggacgct atcaacacgg tgtatccggaagcccgcatc cagctgtgca 28560 tcgtgcatat ggtgcgcaac agcctgcggt tcgtttcctggaaggactac aaagccgtca 28620 cccgcgacct gaaagccatc taccaggccc ccacagaagaagcaggccag caggcgctgg 28680 aagcgttcgc cagcgcctgg gacagtcgct acccgcagataagccggagc tggcaggcaa 28740 actggaccaa cctggcgatg ttcttcgctt acccggcagatatccgcaaa gtcatctaca 28800 caaccaacgc catcgagtcg ctgaacagcg taatccggcatgccatcaaa aaacgcaagg 28860 tgttcccgac ggatgactca gtgaaaaagg tcgtgtggctggcaatccag gcggcttcac 28920 agaaatggac gatgccactg cgggactggc gtatggcaatgagccgcttt attatcgagt 28980 tcggtgaccg cctggacggt cacttctgag aaaaggcatttacacagaat cgtgtacagg 29040 gtcgatttta ctcgttaatt agcgaacaaa ccactcgtcagcagactccc aggtatcttt 29100 cagcgtctcc tgaacaaaag tttttgcaga atctttatcggcggtgcgag taactgaaag 29160 gccatcgttg ctggtggatt tgatgatcac ttctacatcgtcataacgtt tactgacacg 29220 tcgcagcatt tcttgttgca gggcaggaac agaaccttttggcattttgt tgattttggc 29280 tgcggggtat ctgtggggca tatatgggac ataaaaaggcctcaaataaa cagcaaaatc 29340 gagattaggg atttttagaa aaaatacaac catctgaaaaaacttagaaa acacccaaaa 29400 acccacagaa tagtaaaaaa catctatgaa ttatggatttcaagagtcat tcaatctaac 29460 taattgataa ataaaaaatt aacttttcat cgcttcgttatggggcatgg ttggggcaaa 29520 ctcgcttaac tgtgtattta acagcgttac ctgcgcattattattttctg acatccattt 29580 cccgtacacc tgaaacacca tttgcgcatc tgcatggcccatctggtttg caatgaatgc 29640 cgggttagct cctgctgtta acgaccagca ggcataagtgtgtctcgact gatacgattt 29700 gcgatggcgg attccggcac gctttaccgc tgcgtcccacatctgcctta ttgagtcaac 29760 ggtaaaatgg tcgccataat tttttacttt cgctgacacttcaggttgaa aaacaaaggt 29820 acatttttgt ttctctgttc tgccgaattc tctgaggtgaacatcgatga tatgctcttt 29880 gctcagtctc gttagtgcca tctgactccg gagagcgtcgattgcgggct taataaggtg 29940 aatcacacga ttggttcccg cctgtgtttt tggtactgtaaaacggtctt ttgccagatt 30000 tcttctgatt atcattctgc cattgcgcaa gatggttgcggtttttcttc atcattttct 30060 tcttctgttt cctgcttagc agacagttcg cggttaacttcttccaggat atctttttcc 30120 ggtgtatgtc gtgcagcagt gagagtttcc ttgctggggttctcgtgatc agtctccgtt 30180 aagtaggcgt tgatataccc ctgaaggcgt cccgggtagtgataaaactc agggtgagcg 30240 ctccggataa gtgcaaaaat agcggcgcgg gaatagtccagaatacccgg ggttgcgcga 30300 agtgctgcgg accattcttt gaacggactt tctcttttcaggacgatttc ttttgcgcga 30360 cgataaacgc tgcccggaat ttcataaata ttaaaatccatcggaagtgt ggctgctgca 30420 atctccacat ccagcgtatc gaaggtgtgt actaaattcggattgcgatc ggttttgttc 30480 ccgccaccgg catttgcacc ggaagccgta cgggtgatacgcgaaacacg atttcctttc 30540 atccactctt ttgtcagcag accccgatca gtgtagtcagcgtccaggta tgcctcgaaa 30600 aaagcagtta tcagtcccag gtctgaatta ccgggattagggaaaacttt gtcagtgtcg 30660 cgtaccagtt tgtgaaggtc gcgaatctcc agcgggtcgagcagctttgt tttgtgagaa 30720 atggccaagg cagtaacagc cggcagttct tccgcccgtgctatatgtaa tgcctgaagt 30780 tcttcccgtg caacgtgcgt tactggtttt tcgctgccgtgttgcgcaag ccagcgaatg 30840 ggcagctcct gaccagaaac cggcaggagc atattctcctcaatctccgt catgtcttcg 30900 ccgttgacgt tggtattgtc agtactggct ggtttctcctgcacggaggg agagggcgcg 30960 ataaatacca ttgtaatgcc atcttccccg cctttttcgtatcggttgca gaattcggta 31020 tcaaacacgc cttcaggtgg aaggtcattc acaacgggtaaatggacgcg aacgggtttt 31080 ttaaagtcgt cttcatcata atcgttgtca tccattgcggtaatgcagcg ggagatggca 31140 acagataatt tttttgctgt agtccagtaa aaaccacctttaattcccag acgttttctg 31200 actttgtcat tttttgcttc gcaatatagc gcaaattcttctttatcagt gctcattgat 31260 aaacctcatt acagatttaa gggtgaacaa atccctgccattgctggcat ttttaatccg 31320 ttggtatggt gttaatatgg ctggagggtt atccagccggtgtttcgtta ttcaggtaca 31380 gcgatacttt ttttaccggg aggcattcac cagaaattttttgctcgtct cttgcctgga 31440 ggcaggattc tttactggca taaattccgg taatcacattctgtgattca cccgttataa 31500 gaaaaaccgt catcatcagt gcaaatgctg aagtcattgacgttctccga aaataccaag 31560 ttcaagaaga gcaattcggg aaagtatgga attatcattgagcagataag gctcatattt 31620 cctcatgtta atggcatctt cagtaaactc ccggttactgagcagaacac caatatcaaa 31680 acaaccttca gacgtattaa cgtttggtaa taacgtttccattatcgcgt cctcaacaat 31740 gaattttgtg atgcagtgcc tggtgcctcc aggtgacgttaaccagttaa caattaacgc 31800 cggatacaga gaatccaccc ataacactgt ttttggttttaactgttccg cgtgcgctta 31860 gccgcattca ccgcatcaca aaattcactt taaaaagggcggcagagcag ccacggagta 31920 aaactgatac cgccaaacgt caccagaaaa ttgataacagagggcgttgc agcggggttg 31980 tcacttaagc gtatggtcaa cctgacaacc cggtgtcctcaatggggaag gaataacccc 32040 gccatactta ccgccgcgcc atttcgcgga gtgccacaaccggaagcgca cgttcgaaga 32100 aatctaacga caagccttct aagggaaaga rcttcgccgtacgctttcgc gttatgcact 32160 gacttttcag ggaaatatcc tttcagtaaa ctgtcagtaccggattctta tccgtgtccg 32220 gcgcacgacc acacgtgaca gcgtgttggt ctccatttttaacccagaac ctcaatggag 32280 gataaaatgc caaacaaaaa aagaaatccg cttattgaaaaacagattga atgcctggta 32340 aatcaactca ggcaatcagg gttattaaaa actcattcagagttgaggct cacagaatca 32400 gcattcgacg ataaattaaa taatgtcctt tataatggcattattgattt taatcgttct 32460 gttggtcgcc gcggccctgc tggtgtttcc ttataattaccagtcaatcc agagtggacc 32520 gtgttcagcg taaatataac tgtacacatc cagattatatttgtggtctg ttaagaacag 32580 gccgcaaata catgccgaag cttccagtgc agcggctctgttactgaata accatgtagc 32640 aacattccag cgtttttctg catcccagtc tttctcaaggcctgatacca tgaagaaacc 32700 gttagtgttg ccatcaaata attctgtttc caaatttttaagcaatgcct gatggactct 32760 tgccaggtat tccgccggaa tttcgccacg aattctgatgagattgtcat aaacaaacat 32820 gttccccgca tatggcgatt tttctttctt gttttttaaaccagcatcat gagcaaactg 32880 atcaatttct tcttccgttg gtttcgtatt gatgttttgcgctgtcgttt ctgcaatttt 32940 atttgccaca ctctctgagt cgtgtttatt tatagacgcacagaaataca atccggtaaa 33000 cgcatcgcgc acattacgag ccatattatc agtgtcttttttcgttaccg attccaattc 33060 aagttcgttc agacgatgac gaagtgtgtg tgctgcaatctcctggattg aaggaggtaa 33120 atctttaaat tccatcgtca acctcatcag tcagtgtttctggctaacca gcgacgcgcg 33180 ccagcttcag ttttaaacgt tttgcttttg gtatacgtcatcgcggtgaa cgtaccgtcc 33240 tggttgggga acacgccaca taccagagat tcgttgttgccaagattgag cgtatccatg 33300 ttgacctcat ttccccttaa cgccggggta gcggaacaaaaacctgctgc atagttatta 33360 aagttgaacc ctgccgtcat gttcttacgc ctcgggctggctacttaacc cctgaccact 33420 gcctggtaac tcgaagtatt gccctgcatt ctgtggggcggggtgaggga atgaatgaag 33480 tttagaaaaa cgaacattta aggtcaatgt ttttttatcaaaacatttta agcaggcagc 33540 tgttacgcca tcactacgat ggcatacagt taatcaaatagatgaggttg gttaaatatc 33600 ttgttgaatt ttaaagcata ctcccaatat gcaagatagatcatccagca taattgaagg 33660 gtagcgagga ttcgtgggga ctaaaagaat atccggcccttctatctcca gtttgcgaat 33720 gacaggcgtt gtggtccctt tgggtaaggc aaggacaatatttcctggtt gtacgattcg 33780 atcgggatca acaaaaactg ttgaaccatt tgggatggaaactccaccac cagatgtcga 33840 catactgtca ctctctagaa caacagcaaa ggtattggccgggatttctc cgacaagctg 33900 cacacaagag gttattgagg aatttttcat ataatcactccagcttgctg cctgctgaag 33960 tgatagtagc ggaaccgttt ttatcggcgg taaagatagatcaagcgaat cacctgtatt 34020 taactctcct ccattaagaa gccaattttc gtttactttcaatatctttg ccagtgaact 34080 tatgtaacgc gaggacggcg ctcctccacc gttcatccattgacttacgg agccttttga 34140 tgcgccagtg gcattgacaa ggtctttgcc tttcaagtttagcgcatgca tacgttgggt 34200 tatgcgttca gatattgttt gcttgttcat gttttgattttaaaacacag atggttttgt 34260 ttcttgactt tctttggttt tgattattaa acttttggcgttcagtttta tggagcgact 34320 catgaaaaaa tcagaagtat taggctattt tggcggagttgttaaaacag ccgccgctct 34380 aggaacgtca aaaaccacag tcagcatgtg gggggaagacgttccgtgga aatgggcgtt 34440 gctaattcag gcagtcactg ccggggcgct caaatatgagttacacatac cgacggttgt 34500 cattcccgat tctgatcata atccgccttc taaccaaggggggattcatg aaaatcaagc 34560 atgaacacat ccgcatggcg atgaatgcct gggcgcgtcctgatggcgaa aaagttccgg 34620 cagctggaat aacccaggct tattttgagt tgggtatgacgtttcctgaa ctgtacgacg 34680 acagccatcc ggaagccctg gctcgcaata cccagaaaattttccgctgg atagagaaag 34740 acacccctga tgcagttgaa aaaatgcagg ctctgttaccggcgatcgaa aaggcaatgc 34800 cgcctctgct ggtggcccgt atgcgcagtc acagctctgaatattaccgt gagatcgtcg 34860 aacggaggga tcggctggtg aaagatgtgg atgattttgtcgcagcggcg atcgcctggg 34920 gcaccctgac taacagtggg ggtcagcctg gtaatgctgttgtcgtgcat tgaccaacaa 34980 tattcatgcc ggatttcttc cggatgttcg agggtaaagttcggtatcag aagaggtgag 35040 tatggctaat gcctggctca gattgtggca tgacatgccaaatgacccca agtggcgaac 35100 gattgccagg gtatcaggac agccaatcgc aacagtgatggcagtgtata tccatcttct 35160 ggtgagcgcg tcacgaaatg tcacgacatg tcacggcgtgtcactacgtg gtcacattga 35220 tgtcacgacg gaagatttag caagtgcgct tgatgtgacggaagacgtaa ttgattcaat 35280 tttgcatgca atgcaggggc gggttctgga tggtgaccttatttccggat gggaaaaacg 35340 tcaggtgctg aaagaggaca atggtaacgt ttcgcaaacggcaaaatccc cggcagagcg 35400 caagagagcg cagcgggagc gggaaaagct gcggaaacataatgctgatt gtcacgatga 35460 gtcacgacgt gtcacgcatc tgtcacgaca agtcacgacagataaagata cagataaaga 35520 tacagataca gaattaaacc ccacacataa cgcgcgcgagagtattccga ccagtgagtc 35580 gaatggtgcg ccgttgcaga cagccgaacc tgaatacctggacggcytga gcgaaccgat 35640 cgggaaattt tcgatgacta ctgtctggca gccgtcgccggattttcgac aacgggcagc 35700 agtgtggggt atggctctgc ctgagccgga atttacacctgctgagcttg ccgcattccg 35760 ggattactgg atggcggagg ggaaggtttt cacgcaggttcagtgggagc agaaatttgc 35820 ccgccacgtg cagcacgtca gggcacaggt aaaaccagtcagcaaggggg gaagccatgc 35880 agcatcaggt ggcacggcat cacgggcagt tcaggaaatccgggctgcac gcgaacagtg 35940 ggaacgtgac aacggattta tcagcaacgg aaacggcctggaagctgtgg gagctcatgg 36000 gggaggtgta ttcgaaccgc tggactcaga agaacggggccgcaccttcg aagctctgga 36060 ttgcccagat tggtgcgatg actgaacagc aaatccgtctggtgtgccgt cagtgcatgg 36120 accgctgccg ggcgggtgaa acgtggcccc cggacctggctgagtttgtt gcgctgattt 36180 cggagagtgg ggcaaatcca tttggtctta cggtggatgccgtgatggaa gagtaccggc 36240 gctggcgcaa tgaatcctgg cgatacgacg ggagtgataaatacccgtgg ccacagcctg 36300 tgctgtacca catctgcctc gaaatgcgta ccagagggattgagcgccag atgacgcaag 36360 gtgagttaaa acgacttgcg gaacggcaac tgacgaaatgggcaaagcat gttggtaacg 36420 ggatgagtgt tccgccagtg cgacgacaac tggaaggggcgaaacacccg caagggccaa 36480 cgccaattga acggctgaaa caggaatacg aacgccggaaggcagctggt tttatttgaa 36540 tctgagaaac gattttgtcg gaggaaatat taatggaaaccgtatttgac gcactgaaag 36600 cactgaaaaa agcctcttca caggtagtgg catcgcgccttggaatcagc cgcgaagatg 36660 ctgtcaacga actgtggaaa ctgaagcgcc gtggtgaagcggataacaag gggtcgatgt 36720 ggtggctgac tcagactggt gaaagtgaac cagtgtcaccggtaccgaaa gtgacagcgc 36780 aaatgctgac tgaggcgatt gaacatcatg gcccacaaaacggcggatga gctggcactg 36840 atgttcggga ttacctcccg ccgggcgaat tcatcactggccatggcaat cagcaaaggg 36900 cgtctgattc gcgtgaatca gggcggtaaa tttcggtactgcataccggg cgctgattta 36960 ccggcagagc cgaaagccgc atccataacg gaaacggatggtaaagcctt tcctcagcca 37020 gcaggtgttg cgttaccagt cggggaagcg gaaacacaggaagaaataaa aacggaaagt 37080 gtggcggtca cagtgcagtc acagccgtcg ttcaccagaaagcatccgga tggtctgatt 37140 ttaccatcgc tgcatgtggc taaccgcgag ctgcgccgggcaaaaggtca ggttcagaag 37200 tgggagcgag tctgcgccgc gctgcgggag ctgaacaagtgccgggatat tctccgggat 37260 attaccgcca ccagagaaca gcagcggtga gtgggtggaagacgtggtgc cgggcggaaa 37320 tcatgatact ccggcagtgt gcgggaacga tgaaggtaaaaagcgttggc gcacttatcg 37380 gacgaactga agcggcagtg agaacgaagg cacgggagctgggcatcagc atgatgttac 37440 gtggtgattt tcacccgtcg gcaaaatatt ctcagcgtgatattgagctg gcgcggcaac 37500 tgcatcagag aggcatgcaa agaagggaaa ttgccagaaaattaggcatg ccgctgcgca 37560 tagtgaataa ctacgtttat ttcgacagga gggtgtctgcgtgaaaatcc tgtatcagga 37620 ttacggcccg gtggggcagg tggttatcag cagtactgtaatggagtttc ggaagcataa 37680 ccgtgtggtg gatgctgtgc tgttaacctg tccggggatatcggcgagtc gtgcaggtgt 37740 gtttattatg aagacgaaat tatatggcag taaggcgtggataaagaagg cgtatcgtgt 37800 agcgttgcag gaggttaaca gtgagtgaaa ttaaagaaatgccggtagtt cgtgacggat 37860 atggctactg gacacatcct gaatatgaaa aattctgtgatggtcgggaa tatatttcaa 37920 cggaagagtt taacgcctgg atggaggaaa ataatcttcaatacgtcctc tgcttcagag 37980 atgaaggatg tgctgacctt gatgcgtgtg atgctgatatttctgcatgg gaaccggaac 38040 gaccagaggg cgatggctgg tttattggtt ccattcatgatacggaagat ggcccggttt 38100 gtgtctggtt gcgaaataag gctgaagcat aaaggcgataaaccaactaa caactaaata 38160 ctgaagattt aaatcagaaa cgatttttat taaatccttaaccggaggga ttcctgcacc 38220 ctcagaacat caggaggccg cccgaaaggg cggtaagaaatgaaacatta tttagaaaaa 38280 aattacccac gaaagagcag aacaacagag tttctgtttttcattctgtt tatagtgttg 38340 atgataccga tatccccgct attactggtc tggataattggaaggacatt tgaaccagtt 38400 attgagctat ataccgatgt gacatgggaa tcattcagcgcactgcacaa taaaattaat 38460 ccgtataagg aaaactgata tgagcactat taccagagaacgcgcggaga ttaaatcata 38520 catcacaggc ttcctgagcg actcggcgca cgataacaagtcttcagaca gcctgctggc 38580 taatgtgttt cgtatcgcgc tggcatcact ggaagcagagccgatagcaa tggtagtgcc 38640 tgatgaaatg gatttgctta cctgccatct cgacggtgtaactaaaacat atgctgatgg 38700 ctggaacgcc tgccgcgtcg ccatgcttca ggccggaaactttcgggaaa ataagaattc 38760 gtcaaccaac aattttcggg aaatctcgga aacgtcaaccagatctccga taactctgga 38820 tggctggata agctgtactg agcgaatgcc tgaaaagagccagaacgtgc ttatttcgat 38880 gaatatcgat agcgaggctg ggccattaat atattccgcacgctatctcg gaggcacgtt 38940 ccggcgcgga ggtatagcag ttagtccggg taatgatcttaggcaagcaa cccactggat 39000 gtcgctacca gaaccgccgc aggaggtgaa tcaatgacctggcctgaagc attcacaacg 39060 gtaggaattg cgatggcggt ggcgctggtg gtgtattcgatttgccgctg gggataaaaa 39120 cggtttgcgg gaaaaggata gttaagtaga attgctgcgggtgcttgagg ctatctgcyt 39180 cgggcatgaa caccaacggc agatagagaa aagccccagttaacattacg cgtcckgcaa 39240 gacgcttaac attaatctga ggctcaatcc atgctgaacacatgtaggtt agcctcttac 39300 gtgccgaaag gcaaggagaa gcaggctatg aagcagcaaaaggcgatgtt aatcgccctg 39360 atcgtcatct gtttaaccgt catagtgacg gcactggtaacgaggaaaga cctctgcgag 39420 gtgcgaatcc gaaccggtca gacggaggtc gctgtcttcgtagactacga atctgagaag 39480 taagagacca rgcgggggag taatctcccg ccacctyctgatgtgtcagg catcctcaac 39540 gcacccgcgc tttaccatac tgaaaatgct gtttgaatgttcatctctga aagaggacta 39600 tgaatgaaaa aggtattgat tgcagcactt atttccggtgtgtcttttgg cgcttttgca 39660 cagcagggtg gtttccaggg gccagaagca gagcgttcaacagtagcgca ggcaaaagaa 39720 ctgaaggatg atgcatgggt tatccttgaa gggagcatcgttaaaaaagt gggtgatgaa 39780 cgttatgagt ttcgtgacaa tagcgggaca attgtcacggatattgatga cagcgtatgg 39840 gccgggcaga atgtttctcc gaaagacaaa gtaagaattgagggtgaaat tgataaagac 39900 ctgagcagtg ttgaagttga tgtaaaggca ctgaaattattaaagtaacc gcccctgctt 39960 gttaagcccg tcttactgac gggttttctg tttgtacattccggcgtatt gccttacaat 40020 tcgcgcagtc agcctgaaca actgacacct gctgtcaccggagaatccga tgacacaaca 40080 cataaaatcc cacaattctg aagccgaccc ggaaattaagcaggggaggc gttttcgtgc 40140 gcctcagtat ggctggtttc actatctgtt nctgtacgatcgatgargca gatatgcttc 40200 aagaggcgta tctgcgtcgc ggtgtccgtg tggagcggagtctgaacgct gatcgtctga 40260 cctggaccgt ttctgtatat cttcctgttc gtgcacatctgccacggaca catgcctgct 40320 accgtcagcg cgtctggagg taatgtgcgg gtattacttcgacctgttct ggttccggaa 40380 ctcgggctgg tggtccttaa gcccggtcgt gaatcattgccagtttttca tcgcggcagg 40440 gtgctggtgg agccggaacc gaaaaacatg cgggcgctgccatctggagc ggttcctgct 40500 gttcgccagc cgctggcgga agataaatca ctgctgccatttttcagcga tgagcgggtg 40560 attcgtgcag ctggcggcgc tggtgcactg tctgactggttattacgtca cgtgaaatcc 40620 tgccagtggc cacacggcga ttatcatcac agcgaaaccgttattcacag ttacggtgct 40680 ggcgcaatgg tgttgtgctg gcactgcgac aaccagctgcgcgaccagac ctccgaatca 40740 cttgagcaac ttactcaaca aaatctgaca gcctggatgattgacgtcat acgccatgta 40800 atgaatggca cgcaggagcg ggaattatcg ctggctgaattatcctggtg ggcagtctgc 40860 aatcaggtgg tggacgcatt acctgargca gtatcgcgtcgctctctggg attaccggcg 40920 gaaaaaatcc gctccgtata ccgtgaaagc gacatcataccgggagaaca gaccgccacc 40980 agcatactga agcagcgcac aaaaaatatt gcgctaccgcctcacaccca ccagcaacag 41040 aacccaccac aggaaaagac ggtggtcagc attgccgttgatccggagtc tccggaatcc 41100 ttcatgaaac gacctaaacg tcgccgctgg gtaaatgagaaatacacacg ctgggtaaag 41160 acacagccgt gtgcgtgttg tggtaagcca gcggacgatcctcatcatct gattggtcat 41220 ggtcagggcg gaatgggaac aaaatcccac gatattttcacgctaccgct gtgtcgggag 41280 catcacaacg agcttcatgc ggatccgctg gcgttcgaagaaaagcatgg ttcccaggtt 41340 gatttaattt ttcgttttct tgatcacgcc tttgcaaccggcgtgctcgg gtaaaagagg 41400 ttactgatgc gtatagagtt tgttttgcct tacccgccgacggtgaacac ctactggcga 41460 cgtcgtggca gcacatattt tgtatcaaaa gccggtgagcgttatcgccg ggatgtggca 41520 cttattgttc gccagcagcg gctgaaatta aacctgtccggaaggctggc gataaagatt 41580 attgcagagc caccggataa gcgccgtcgt gacctggacaatatcctgaa agcaccactg 41640 gatgcgctga cgcatgccgg acttctcata gacgacgagcagtttgatga aatcaatatt 41700 gtgcgcggct agctcgtttc tggtgggcgg ctgggcgtgaagatttacaa aattgaaagt 41760 gagtgagcat aaatatgata tacccggaaa ttacaggcaaaagcggtgag catttacgcc 41820 tgaaaacgct ggaaagtgtc tggatccagg ggaaactgcgtatgtggggg cgttggtcgt 41880 atattggcga cggtaagacg ggaaatatgt tcaaccaattactgacctct aaaaagctga 41940 caaaaacggc aattaacgag gcgctccgga ggatgaaaaaagcgggtctg gacaaacctg 42000 aacttgaggc ttttttgcgg gatatgatca acggcaatcaaaaaagctgg ctggcacatt 42060 gtaccgattc agaggcgtta ataatcgata gggttattggtgaagtactg gcaggttatc 42120 ccgggctgct caatgttctg agtcagcgtt atgtggggcgggggatgact aagcgcaaaa 42180 tggctgaatt gctgaatgat gcacatccgg aatggagtttaagaacctgt gaaagacgca 42240 ttgagcattg gctaaaggtg gcagaattta ttttgtacaaaccaatggtt atggcttttg 42300 gtatagagaa aaaagttatt gctttttgac gtaaaaactgcttcaattcc ggtaagcttc 42360 gcaaagctgt accgcgaggc gaatagcaga catggacatttgaaagagcc cgctttttgc 42420 gggttttttt atgactgaaa aacggcacgg ggcgttaaacgcgctggtgg ttgctaatac 42480 cggtctttca acttgctggc tttttcgaca agagttattggtatgtcacg ttaaccggaa 42540 aagggaaaaa gacatgctaa aacagcagga tatgacagaaaccgccagag tggtgtttaa 42600 tgaattaagc gttaccgaac cggcgacagt cggggagattgcgcagaata cttacctttc 42660 acgcgaacgc tgccagttaa tactgaccca gctggttatggcgggtctgg cagactatca 42720 gtgcggttgt tacagacgca ttcagtcctg aaggctttttatttgtggta aatgggcggc 42780 tggtgggggg ggcggcacct gtcagtcctt tgcttatgtgttgatgataa tttacctttt 42840 ggggctataa ttgagctaac caattgctaa tgaaagtaaaattataatgg ctgttgtctg 42900 ttcagttatc atggtttgct ccccaattaa tatttttcttgaaaaggata cgttgtcact 42960 taagccaggc tcagtcgttc tggccaccaa atgcatcagggagcttttcc ttatgcatta 43020 tggcaaagtt aaaattgtcg atataagcsa atccgtcgtaagtcaatatc tggaaagtca 43080 gcataagctg acgaggactc gtctgactga cattccgctttacctgttrc tggaacccaa 43140 caaycctgcg ttggctgcgg ctttaattac cagccagggattttccggag aggtcacgga 43200 tatgtttctt atgatggcct gcctgtctct gtttgaaacagatgaacgga tgtcattgtt 43260 tttaagtgga tgtttatcca gcataagtgc caaagtcagggcgataattc agacagatat 43320 atcagcaagc tggacgcttg gtgcgattgc tctacagttgcatatgagtg agagtttgtt 43380 aaagacaaaa ctgaaaaatg aagggggcat gttcagtcgcttgttgctgg aagagcggat 43440 gcgtgttgct gtaaatatgt tatgttcccg gtatggatatggacaggctg tagcagaaaa 43500 atgtggttat tcaagccggt cctactttat ttctgtatttcaccgctatt atggcttccc 43560 gccagacaga tatgtatcca ggcaagggct tgattattgattttcatctg attattattt 43620 tttggctcgg ccctttagct cagtggtgag agcgagcgactcataatcgc caggtcgctg 43680 gttcaaatcc agcaagggcc accatcacaa accgccattagcttatcagg aagagcagac 43740 gacaccataa cagggttgtt ggtgcggggc cgggtccccgatggcggtcc attatcggta 43800 ttctgcgttg ttagctcagc cggacagagc aattgccttctaagcaatcg gtcagtggtt 43860 sgactccact acaacgcgcc acacttattt tccaggctcgcttcggcggg ccttttttgt 43920 atctgcgcca cgcccggcgc ataccaacca cagagcctttcgggggtgag cttacggagt 43980 ggtcagtgtg actttctctg tgggcagatc gctcccgggcgttggctcac ccacccaaag 44040 gaacgtcacg atgtttggaa tcttcaaaaa gaaaacccgcagagcggcaa cggaaattaa 44100 aaagtttgaa aaacgcgatc tggcacaggt ggtgattaacgccgcatacc tggtggcctg 44160 tgcagatggt gaatgtgagg catccgagaa agcgaagatcgaacaggtac tgcggtaatc 44220 agcctgcgct gtacgcgttt acgtcagaaa ttaatgcgattagcgcaacc attatcggtc 44280 agctggatac gaactttaaa actggtcgtc gtgcggcgttacgtgagaty gaggatgtga 44340 aacacgatac gcgtgaagcg gaagatgtgc tggatgtggcggtggccatt gcggaggcag 44400 acggcgaaat tgagccggaa gagcgcaagg tgctggaagagattgccggt gttctgggtc 44460 ttcgtctgga gaatcacctg tgacggtaaa actgcgcctgrctgtggctg cactcctgct 44520 gtttctggtg gtgatggtgg atttcaccag cagaatcatgtcggtgctgg cggatggggt 44580 gctggtctgc ggcattgtgg tattgctgtg gccggtgataaaaagaaaca gcctgcataa 44640 tgcttgattt ttttgtttgc tgtttattaa aaacacttctgcatggtgaa tccccctgtg 44700 cggaggggcg atcagcaasm aggtatatgg gataatcgcggattcaggtg ctgatactga 44760 attcaccggg aggcacccgg caccatgcaa gaaaaagaatgtgcatgcaa acatgcccct 44820 ctccggaggg gcttttttat gggtaaaaaa tgcccgaatgggttcgggca atagcatgag 44880 atactgatat tgttgtgttg ttatcgtgtg gattttaaccagggtttatc aggctgcgca 44940 actgcgtggc cttttttcat ttcttgggct gtagtccccgagtgtcattc aggcttccgg 45000 actacagcca gcccactcca tatctgattt aatmcactatcccggccggg aggaataatg 45060 acatttaaac attatgatgt tgtcagggcg gcgtcgccgtcagaccttgc ggaaaagctg 45120 acacacaaac tgaaagaggg ctggcagcca tacggcggaccggttgccat tacgccgtac 45180 acactgatgc aggcggtggc tattgaagga gagccacaggtcggcccttc atctgagccg 45240 gattggtact acgtcatcgt actggccggg cagtccaatgccatggctta cggtgaaggg 45300 cttccgctgc cggattcata cgatgctccg gatccgcgcattaaacagct ggcgcgccgc 45360 agtacagtga cgccgggcgg ggctgcctgc agatataacgatattattcc ggctgaccac 45420 tgtctgcatg atgtgcagga tatgagtacg ctgaatcatccgarggctga cctgagcaaa 45480 gggcagtacg gctgtgtcgg ccagggktta catattgccaaaaaactgct cccgtatatc 45540 ccgaataacg cggggatcct gctggtacca tgctgtcgtggtggttcggc atttacccag 45600 ggcgcggagg ggacattcag cgagtccacg ggggccagycaggattcggc acgctggggg 45660 gtgggcaagc cgttatatca ggatctgatt tcccgcacaaaagcggcatt gcagaaaaat 45720 cccaaaaacg ttctgctggc cgtctgctgg atgcagggwgagtttgacat gagcgccgcc 45780 acccacgcac agcaacctgc gctgtttaca gccatgctgrcacagtttcg tgctgacctc 45840 tccgtgttta acgcgcagtg ccatggtggc agtgctgcagatgtgccgtg gatttgtggt 45900 gayacgacgt attactggaa aaatachtac gsyacccagtacracaccrt gttacggsgs 45960 gtayaaaaac argggagagt gagggcgttt attttgtgcccttcatgaca gacggtaacg 46020 gcgtcaatac cgccactaac gcgccggcag aagatccggatattccggca tcaggatatt 46080 acggtgcggc atcgagaacg aatggaaacc aggtatcatcaaaccgcccg acacatttca 46140 gttcatgggc gcgcaggagc attattccgg atcgtmtggcaaccgctatt ctgaacgcag 46200 ccgggcgcac ctcmgccttc atcagtggta aggcaccggaaatcaaaccc tcgcccggcg 46260 gcaacacgcc atcgggtccg tctgcagata cgtccgttcgcacaatctcc ctgctgccgg 46320 cagccggara ggctgctgcg cagggctgga gcattaaggatggcggaatt cagttgtcag 46380 atggtgtatt taagatcacc aggcagagca ataaaacctggtccctgacg catccggtgg 46440 atgacgcaat taccctgctg acacagggcg gcagactgaactgtaagttc cgcctgtcag 46500 gcgcactgac caacaatcag ttcgggctgg ggatttatctgtatacggat gctcccgttc 46560 ctgatggtgt ggcgatgacg ggtaccggta atccgttcctgatgtcgtac ttcactcaga 46620 ccactgacgg cagagtgaat ctgatgcatc acaggaaagccggaaacacg aagctggggg 46680 agttcggcga ttacggtaac gactggcaga cgctggagctggtgttcacc gccggcagtg 46740 ccacggttac tccgaaactg aatggagtgg ctggcccggcattccaggtt ataaaagaca 46800 gtctgacact gggactgaat gcgctgacgc tgacggatgttacaaaaaat gcagcgtatg 46860 gcgttgagat agaaagtctg gtgctggaga taaatgcaccggcagcataa taaaaaaaga 46920 gccagcgact gacctgaaag aagacgctgg ctaaaaggccttatatgttt gtagagactt 46980 atttttcaca gacagcaatg atgcctgtca atatattatcaatatgcgga ttgaaccgcc 47040 ccggaaatcc tggagactaa actccctgag aaagaggtaaacaggatgac taaaaatact 47100 cgtttttccc ccgaagtccg tcagcgggcg attcgtatggttctggaaag tcaggatgaa 47160 tatgactcac agtgggcggc aatttgttcc attgccccaaagattggctg tacgccggag 47220 actctgcgtg tctgggttcg ccagcatgag cgggataccgggggcggtga tggtgggctc 47280 accagcgctg aacgtcagcg tctgaaagag ctggaacgtgaaaatcgtga actgcgccgc 47340 agtaacgata tccttcgcca ggcttccgct tattttgcgaaggcggagtt cgaccgcctc 47400 tggaaaaaat gatgccactg ctggataagc tgcgtgagcagtacggggtc ggaccggtat 47460 gcagcgaact gcatattgcc ccgtcaacgt attaccattgtcagcaacag cgacatcatc 47520 cggataaacg cagtgcccgt gcgcagcacg acgactggctgaagagagag atacagcgcg 47580 tatacgatga aaatcatcag gtgtacggtg tgcgtaaagtctggcgtcag ttgttacggg 47640 aaggaatcag ggtggccaga tgtacagtgg cacgtctcatggcggttatg ggacttgccg 47700 gtgttctccg gggtaaaaag gtccgtacga ccrtcagccggaaagccgtt gccgcaggcg 47760 accgcgtaaa ccgtcagttc gtggcagaac gacctgaccagctgtgggtg gctgatttta 47820 cttacgtcag cacatggcag ggcttcgtct atgtggcgttcatcattgat gtgtttgccg 47880 gatacatcgt ggggtggcgg gtctcatcgt ctatggaaacgacattcgtg ctgaatgcgc 47940 tggagcaggc gttgtgggcc cgtcgtccgt ctggcaccatccatcacagc gataaaggct 48000 ctcagtatgt gtcactggcc tatacggagc gactaaaagaagccggatta ctggcatcaa 48060 cagggagtac aggcgactcg tatgacaacg cgatggctgagagcatcaat ggtctttaca 48120 aagcggaggt aatacaccgt aagagctgga aaaaccgtgcagaagtggaa ctggccacac 48180 taacgtgggt ggactggtat aacaatcgac gattgctgggaaggctgggc catactcctc 48240 cggcagaagc agaaaaagct tattatgctt ccatcggaaacgatgatctg gcagcctgag 48300 ttcacagata aaacactctc caggaaaccc ggggcggttcacccatttca ttcagcagac 48360 gttcacgggc catttccagc gaacagtcag gatcagccacgcactgtgcc tgaagcgtct 48420 gatagcgacc gccgaacatg gcaaacagat cgttaatgcctgacatgcgg gctttctgct 48480 cagccataac gcgggcgcga atggtcgcct catcagacactgccggtacc ggtgatggtt 48540 ctgttacngc cggtgcaggg attgtcactg tggtatcacgcggggcactg ttgcgtggcg 48600 gagtaatcat gtttcggatg gattccggca tctttttaaattcctctgta cgttttgact 48660 gaatacatgc cattgcctca acagcgggtg tcacctggtcagcaaatccg tgtgccagac 48720 attcggcacc ggacatccag gtttcatccg ccagcatggcggcaatttca tcggtggttt 48780 ttccggtttt ctgcgcatag gctggcaaca gtaccgattcgactttatcc agcaaatcgg 48840 cataactgcg catatcctca gcatccccgc cactgaatccccatggctta tggatcatca 48900 tgaaggcatt ttccggcata atgaccgtat caccggccatcgcaatcaca gatgccatcg 48960 aggcggcaac gccatccaca tacacggtaa tggtcgccccctgatttttc agggcattaa 49020 aaatggcgat gccttcaaag acatcgccac ccggtgaattgatattggag attaatgtgg 49080 gtgatatcac ccagtgcatt cagttngctg acaaactgcttcgcggtaac tccccagaaa 49140 ccaatctcgt cataaatata aatatccgcg tacccggccccccagcttgc atctgaacca 49200 ggatttattc ttcatgctgg ctttcggtgt cgcgctgattgtcgtcagtc ggcntgtnct 49260 ctttgtgtga gggncagata aanacagccc agtttgtgtttatcattnaa ccttgggnga 49320 gttgactcat cggattggcc cacggacgca ccagtcagattctgcgttgc ccacccggat 49380 ctgaattctc caggcttcag cttccttaac sgggtcgatccacggcatca ccggaccgga 49440 atacgtcgcg ttatatagcg ttttcatctc cacatccgccggaattttca gsrgacctgc 49500 cgcaaccacc atattcagcc atgtccggta caccgggcgggttaccgcgc caataaaaca 49560 gtcctgcagg atcaggtaac catccgtgga ctcgaccagctcctgccgct gggcgctgta 49620 ggtgccgtta tagttacgcg ccgcactgga aaaactcagacgactgcctg ctgccactgc 49680 acgcaactgg ccgttgcgga aagtttcaag gttgggattgggacggtcag atttgaccat 49740 gccgatatcc tcgcccttgc gcaaatcgtc ataaataatacccggggtga tatggacttc 49800 ccgctcggtc tctttgatcc ccnggatctt catagtcctgtccgtcacct ttacggatat 49860 acagtcccag cgccgcagca atacmgcgcc gctgtcagttccgcatcctc atactcctta 49920 agggcactga tccgcatcag cacccccgat aacatggatgagcctcgcgt ctgatgcaga 49980 cgacgagtga acttcaggtg gatcattttt ccggcagcgatttctttcgt atcactctgs 50040 cggccgctga ccggataatt tttataaacc agatattttttcggtcttcc ccactcatca 50100 aagaaaaacc cccctgattc agttccggcg gattcaatcagtgcgcatgg gaaacaaaat 50160 ccggnctcca tccgcctcaa gccagaaatg gcactcccgccgtccgttcc agaccgtttc 50220 ccgcaccact gaccatctgc gcaaacactt caccatcccgcagccaggtc cgcagcagta 50280 aacgttcaag cacaggacgg gtatactgcc ctgtcacatccggactcacg gaccattcag 50340 cccacaaacg gcggatatcc gcagccagct cagccgccatttccccgttt tttygtaatg 50400 gctgaggctc cacaataatt cccctggcac caatcacccgctcttccagc ttgtcaaaca 50460 caccaatcac caggtcatga ttgatatcca gaaaacgggcctgctcccgc agggaaaccg 50520 caccgtattt actgagctga tcagcagagc gattttcccgccgggcttta tgtgtccggg 50580 tcggtttcac cgcctcatag gccatgatta acgcccttgaacgcagtctg gctgctttcc 50640 acccggggga aaacacgccg atcacatcat caataattgccattaaaacc tcgccagttt 50700 aaatcccggt tttccccgcc tgcggctcac catcgcggcaagcctgcgtt cccactcctg 50760 acgtccggcg cggatctgag aaaggctttc cagcgtcagttgctgcccgt tgaagatgac 50820 agactttccc tccagtacgg ccatttccgc ttcacggtaccgctgtatca tttctctggc 50880 ttcttctgtg ctcacaacca gcctcctgat gttatccatggattatcttc cgcacgctcc 50940 gtccgcagtt ttttcttccg gcgacggcgt ttttctgccccggccgtcag ttccggggat 51000 accgtttcac cagaacgctc ctgcgggaag acgagccacgtttcccgctg tgcccagtcc 51060 ggtgcggagg gccagcggat cttttcgtaa ccatgcagaacggcaagcgc atccgcataa 51120 accarcaggt caaacgcttc gttagcgccc ctgcccggttttcgccattt tccgtcactg 51180 ccgcgctctt cataggtcag ctcatcgtaa aaccaccgccccagccagtc gggaaagtgg 51240 atatagttcg gccctggtgt gtcacgccac agggcattatttacacgatc cttaaacgca 51300 tccgtctgaa ccagccacag cgcgacatcg ccactggctctggcacggcg ggcacttctg 51360 ccggtattat ccgggaaggt tcggttaatc agcctgtcacgggcgaagtc catccccctt 51420 gaacagaaac accctgttgc ccagtccgtc actccggcaacgacgccaga aacgataggc 51480 gttatctgtc accccggctt cccctcccgt atccaccgccatggccatca gacgcatgcg 51540 cacatccgga tcagaagcca gcggccatgt tttatggaacacatccgtca gcaacaaatc 51600 ccagtcctcc ggatrtgccg ccggatcaac cggcagactttcaccgttgg gactgcagcg 51660 cagtgaatgc cggatgttgt agcgatcaac aatccagcgttccccctgct ctccgtatcc 51720 ggtgatctgc acaacaaaac ggcgattttt accgccctgtacgtcaaccg ttgcctcaat 51780 aaaacgcaca ccatccggca cagatcgccg gggaaacggctcggcacgct gttcaagcag 51840 ttcactttta cgctgttccg tggctgaacg gggcagatagggtcgtccga tatcggtgtt 51900 caccaccgct ttcagggtct cttcactgcc ggttcgctcatactcttctt ctgccgccag 51960 cagtttaaaa atcagttgtt cccaggtctg aaacgccgcagctggcccct ccatccaaaa 52020 tgacgcaatc cgggagtttc gtggcgttcc ggtgatactgccgtccgccg ccgcccgttc 52080 accttcacga agccagatcc cctggttatt cagttcgcgtttctgctcag gggcaatcag 52140 cccgcgacaa tgcggacaca tcagacgggc agcctgaccggcagccacaa aatccgggtt 52200 attccggtat ccggtcatgt tatccatcac cggctgaaaatattccccgc agtgcggaca 52260 cggccagtac caccggcggc ggtctccccg gttatacagtgacaggatcc ccgttgttgg 52320 cggtgcctca tgtgcgccac cacaacgcca tttggtatcggtgatatccc gccccggtga 52380 actctcgacc agggtcatcc ccgaggacat aaaggtggtggtacgctttg aggccagcgt 52440 gaaggcatcc ccttccccgt ccacattttc agggaaacggtcataatccg tcagcgccac 52500 acgacggtaa tccgaagagg aaaagacggt gatcgacggccagccaatct tcaggaagga 52560 gccgtcaaga aacattttat cgtggacgtt gttgtcattacgggaaggac tgaggcgctt 52620 gctgacctcc ggactgtggc gaaacgtcct ggaaagacgcgttctggaat gctcacgcgc 52680 cttcgtctca gtcatctgca ccaccagcat atccgccggatcacagatga tgccgtacac 52740 aatccagcca tcaatcagcc cttcggtttt cccggttcgcgcaggtccca caaacaccac 52800 cgcgtcatat tcacgggctg ataatgtatt aatggggtcaatcatatagg gcgtcagcga 52860 tgactcccac ggaccggaag tattggctcc ccgtggaacccgcatataac gcctgatggc 52920 ttccgctact ggtaaccggc tgggtgggcg aaacagcgaggccacttcgc gccagatatc 52980 ggatgcgcgg ctatggctct cgttcacctg attcacatatcggcctcatc acaacagtca 53040 atgactgcct tttccagtgt gtcgcggatc tcatcaaccacaatctgtac ttcattcagt 53100 tgtgatgcag tccaccccct gtccctctcc agccggtcaggccaggtttc cagtacctga 53160 actatcgctt tcaccacgac agaaaaggac cgcctgacatcactgactgg cacaagctga 53220 acagtttcat gctgaaattt aagacgctcg cgctcggactgataccatgc cttacgagcg 53280 tgaggatcca tatcctcatc ttcggaagat ggtggtttttccagcaacga agttatcaaa 53340 tccgtcagga gatacagttt tttcttttca ttactgcctggtgcaagagg aacatccgcc 53400 attctggcgg caacagtctg ccggtgcaga cctgaaagggctgccagttg attaatattt 53460 aacttcatat ttttcagctc gccgtccatt tacatccctccacataaacc gcagaacaga 53520 agtgactctg tttttttgta aagaaatgcc gccatataaagatgtcgaac aaaaaacaac 53580 cacaatcatc atctttttaa tactaacagc attaaaaacaacaagttacc atcatgatga 53640 tgatgacgat aaaatcacaa aaatgcgcct ttttccgcgcccgcccgccc cgtgttcagg 53700 cccaccccac caggaggacc cgcaaaatga taatggttatcatttgcaac aaaatccagt 53760 ttcttccacc atcgcaccgg actggcgact atgaggggacaacaccgcgc tccgttaacg 53820 cggtaaaccc cggtgtgtat cgtttttgat tatccccgcacactctcgca gaggagtctc 53880 cctgtctggc tgcggtctct gttaatgcag gaatacggtgacgatacggc gcatcagcaa 53940 aacttagttc aggcactgag tgcggatata gtcctgtgccccttccagct gcttctgcat 54000 tgtcatcaac cgttctctga ggatgaaata atcccgttcagcggtgtctg ccagtcgggg 54060 gccggttgca ttatccacgc cggaggtgcc ggtggcttcacgcacggtac cggagcaggt 54120 ggcgttgatc cgcaggcgct tacgaccagc ggcaacatcagcacgcagag tttcattttc 54180 agctctcgca tcggctaatt ccctcgagta tctggcatcaagtgcagcga catcacgctg 54240 gcgtatctgc atatcagtaa ttgtcgcgtt cgccagctccagctcactgg cttttttatc 54300 gcgctgctct ttgtagatga tggcgtgatc acggtaatgattcagcccca gactaagcgc 54360 accacaggcc accagcagga caatgataac cacacacagaacacggttca tatcaccacc 54420 aacggattgc ccagaccaga acagcaatgg ccacaatacgaatggcaaaa gctgccgctc 54480 ttgttaaatc cagactggct ggcgtctcca cttcaatgcctttcataatg gacaacctca 54540 gaaagaatct tttatacttc ctcacaggga aagtacctccctacccataa tttctccctt 54600 gccttactca aggtcagaaa acacaaaacc ccgcttgctgccaacaaacg gggtttttac 54660 ttttattcac ttaggtttta ccagttttca ggatttcgtgttatccaccc gcgttggcca 54720 acgtcatttt tcaggaaaat attctgctat ctgtcgatgtcccagcacgc cagcgcgctc 54780 tcctggtcac gccgtgagac ctgaccgtag caattatttgaacggatacg gcagtctctg 54840 ccaccgtcct taatccacca gcgaatcgcc tcacaggcaccttttcgatc gcctgcatta 54900 attcgtttat aaaacgtcga cgggaagcac ttaccggggccaatgttgta cggacagaat 54960 gacgcgatcc ccgctttctg gggttcggtc agcggcacccggatgttttt ctccacccat 55020 gccagcgcct tgtcacgttc gatggcatta acccggtcgcatttttcctt tgacagcttc 55080 atgccaggaa taacaggctt accatccacc agaatggcaccacggcagat ggtccagatc 55140 cccgcaccat cacggtatgc cgtggtgtgg ttaccttccttttcatccag aaactggtcg 55200 aggatttcag gcgcagaagc acctgcacca atcagcgccagaacggcagc cgacaggccg 55260 tattttattt tttcgttcat ggggatttat cgatttctaatcccttgata tgttaggtat 55320 ataatccaac actcatggtc gctctcataa acatatcccttgagacgcag cagattacaa 55380 caaatgaagc catataaatg aacagtaaag aaagtttgcgcagaagattt ttacaactaa 55440 tgacagaaaa cgttaaatca gagttacttc ttctgatggcagataataac gaagcaacaa 55500 gcagcattct tgcagaccct tacggtaaga tctcacataaaacgctggat attattacca 55560 caacattaac accgctgatg cttcaacggc tgaaacataatatcaacgca tgggttaatg 55620 aagaattaag tcctccctgc ttatgggatt ctcgttacgcatgtcagcaa aaaatgcgaa 55680 ttttcaactt actatcacca aagctcaggt agccataaaatcctgccctt catggcatac 55740 aggatttcaa tggaatcaca atgaccaact cttgcacagctgtatccctg actccccgac 55800 aactcagatt ttcagtatct gctgctatct aaagagaaagcgcacaaatg caagggtctt 55860 tcatcacgtc ctgttattga ttgcctgtga ccttttcttacctcatggaa cgttttttca 55920 gttagaaata ttcattttac aaccagttcg tattgtttattcatcgacta ctctccccgc 55980 gccaccttac gacggtcctc tctgattttg aaatacaggttagtcagata cgtcagcagg 56040 ccaaacagca gacttcccag cactcctatt gccacccactgggacggaga gactttgtcc 56100 agcagctgca gtaaccagta ttccgtcccc accgctgacgtggtgtatga cacacctgtt 56160 gtgatttttt ccatctgatg tatgtctccg tcaccgccgacagaaaatga aagtaaagaa 56220 aaacaaaaaa gccgccagtg tcacccactg acggccaactccgggagccg tgattatggc 56280 attcaggctc tgctaaaaat gccagataac attccggcctcccctgattc aggttataaa 56340 tgacacaata tcttgacaac acccgtcact gtctgtcagaaaatataccg ccaggcataa 56400 gtatcatgtg aaatccaact atccttctga gccagcacctctccaccgaa agtcagtgct 56460 ggctgttttt ttccttaata aagcatctgt aactggttgttgttctgcgg gttctgttct 56520 tcgttgacat gaggttgccc cgtattcagt gtcgctgatttgtattgtct gaagttgttt 56580 ttacgttaag ttgatgcaga tcaattaata cgatacctgcgtcataatta attatttgac 56640 gtggtttgat ggcgtagatg cacgttgtga catgtagatgataattatta tcattttgcg 56700 ggtcctttcc ggcgatccga caggttacgg ggcggcgacctcgcgggttt tcgctattta 56760 tgaaaatttt ccgggatcca tgtccggttt ctcttcaagttaactatatg aaaaatataa 56820 aaacaggtct tctgtgaacc ggacatgaac aaaaaacagacatgtaaacc ggacatgacc 56880 ggttttgttg tgattgtgag gtgagagttt ttgcgaggtgaggagtggct acgcagactg 56940 aagttgccag gcatttaagt ctgaccgatc gccagcttcgcagattgcag aaattgccgg 57000 gtgccccgat atcgaataag cgagggcaac tggatctggatgcctggcgc gatttttaca 57060 tatcgtatct gaggagaagt aaaaacgatg tgcctgatggcgatagcgaa gacgactatg 57120 aggagaaatt gcttattgcc agatgggaac tgacagcagaacaggctgtt acacagcagt 57180 taaaaaagcg tacagcctga accgtctggt cagaatctgacgaattagac aaagtggtgt 57240 ccaccaaata agtagtggga accaaagtat cagatatgcagaaaaatgtg actcccggca 57300 ggcgaaaagg ctgccctaat tatcctcccg aatttaaacagcagctcgtt gctgcctcct 57360 gtgaacccgg gatatccatc tcaaaacttg ctcttgaaaatggcattaac gccaatctgt 57420 tgttcaaatg gcgacaacaa tggcgcgagg gaaagctgctattaccttct tcagagagcc 57480 cccagctact tcctgtgact ctcgatgcag ctgccgaacagccagaatcg ctcgcagagg 57540 acccggaaac cctcagtatc agctgttagg taacgttccggcacgggccg ctccgcttca 57600 atggcaatgt cagcgaaaag ctcctgactc tgctgatacaggaactgaag cgatgatccc 57660 gttaccttcc gggaccaaaa tttggctggt tgccggtatcaccgatatga gaaatggctt 57720 caacggcctg gctgcgaaag tacaaacggc gctgaaagacgatcccatgt ccggccatgt 57780 tttcattttc cggggccgca gcggcagtca ggttaaactgctgtggtcca ccggtgacgg 57840 actgtgcctc ctgaccaaac ggctggagcg tgggcgcttcgcctggccgt cagcccgtga 57900 tggcaaagtg ttccttacgc aggcgcagct ggcgatgctgctggaaggta tcgactggcg 57960 acagcctaag cggctgctga cctccctgac catgctgtaaatctctttat cctggttgtc 58020 acagaataag cccggtaaaa tacgggctta tgaacgacatctcttctgac gacatcttcc 58080 tgctgaaaca gcgcctggcc gaacaggaag cgctgatccacgccctgcag gaaaagctga 58140 gcaaccggga gcgcgaaata gaccatctgc aggcgcagctggataaactc cgccggatga 58200 acttcggcag tcgttccgaa aaagtctccc gccgtatcgcacaaatggaa gccgatctga 58260 accggcttca gaaagagagc gatacgctga ctggtagggtgtatgacccg gcagtacagc 58320 gtccgttgcg tcagacccgc acccgtaagc cgttccctgaatcactaccc cgtgacgaaa 58380 agcgactgtt gcctgcggcg ccgtgctgcc cgaactgcggcggttcactg agctatctgg 58440 gcgaggatac cgccgaacag ctggagttga tgcgtagcgccttccgggtt atccggacgg 58500 tacgggaaaa acatgcctgt actcagtgcg atgccatcgtgcaggcacct gcaccttcgc 58560 ggcccatcga gcggggtatc gccggaccgg ggctgctggcccgcgtgctg acctcgaagt 58620 atgcagagca caccccgctg tatcgccagt cagaaatatacggccggcaa ggtgtggagc 58680 tgaggcgttc actgctgtcg ggctgggtgg atgcatgctgccggctgctg tctccgctgg 58740 aagaggcgct tcatggctat gtcatgactg acggcaaactccatgccgat gataccccgg 58800 tccaggtact gctgccgggt aataagaaga cgaagaccgggcggttgtgg gcgtatgttc 58860 gtgatgaccg caatgcaggg tcagcgttgg cacctgcagtgtggttcgct tacagcccgg 58920 acagaaaagg catccatccg cagactcatc ttgcctgcttcagcggtgtg ctgcaagcgg 58980 atgcgtacgc cgggttcaac gagctgtatc gcaatggtgggataacggaa gctgcctgct 59040 gggctcatgc ccgccgaaag atccacgatg tgcacgtccgcatcccgtca gcactgacgg 59100 aagaagccct ggagcagatc ggtcagttgt acgccatagaggcggatata aggggaatgc 59160 cggcagagca gcggcttgct gaacgtcagc gaaaaacgaaaccgttgttg aaatccctgg 59220 aaagctggtt gcgtgaaaag atgaagaccc tgtcgcgacactcagagttg gcgaaggcgt 59280 tcgcgtacgc acttaaccag tggccggcac tgacgtactatgcgaacgat ggctgggtgg 59340 aaatcgacaa caacatcgct gaaaatgccc tgcgggcggtcagtctgggt cgtaaaaact 59400 tcctgttctt cggctctgat catggtggtg agcggggagcgctactgtac agcctgatcg 59460 ggacgtgcaa actgaatgac gtggatccag aaagctaccttcgccatgtg cttggcgtca 59520 tagcagactg gccggtcaac cgggtcagcg aactgcttccgtggcgcata gcactgccag 59580 ctgaataaca catccccgtc aatacggccc tcgctgtacgcttacttaaa aaatgaggtg 59640 tcaaaaggaa aactgattga caccgggttc tgtatttttgccctcagtaa gctggcaatg 59700 gcgttatcca gtacgcttga ttccatccct ttatccatgcagcgacagtt tcctgattta 59760 acaccgcgcc atcttgacca tctgaaaacc cttattgctaagggggcaaa tcagtgtgcg 59820 cgggcagggg ataaattacc ggatttactt gatgaatatatcagagcaac aactgaataa 59880 tatgatgagc gctgtcacaa ctgcattaca gcccctgataagggcattgc cggtgacgcc 59940 agttgaatgg gctgatcaaa attattatct gcctaaagaatcttcatatg gtgagggcga 60000 atggaaaacg ctgccattcc agatcgccat catgaacagtatggggaatg atcagatccg 60060 cactgttaat ctgattaaat ctgcccgtgt tggctatacaaagatgttgc tggggggggg 60120 cgggtatttt attgagcata aatcccgaaa cagtctgctttttcagccca cggattctgc 60180 cgctgaagat tttatgaagt ctcacgtgga ggcgacgattcggaacgtgc catgcctgaa 60240 agacctttcc ccatggctgg gtcgtaaaca tcgtgacaatactctcacgc tgaaacgctt 60300 ttcatcgggc gtcggtttct ggtgcctggg cggcgctgccgccaaaaact accgtgaaaa 60360 atccgttgac gtggtctgct atgacgaact ttcctcgttcgagccggatg tcgaaaaaga 60420 gggctcgcca accctgctgg gggataagcg tattgaggggtcggtgtggc caaaatccat 60480 tcgcggctcg acgcctaaaa tcaaaggcac ctgccagatcgaaaaagccg ctaacgagtc 60540 ggcgcatttt atgcgttttt atgtgccctg cccgcactgtggggaggcgc agtatctgaa 60600 atttggcgat gagtccacgc cttttgggct taaatgggagaaggacagcc ctgaaagtgt 60660 tttctacctc tgtgaacatc atggctgcgt gatccatcagtctgaactgg accagagcaa 60720 cgggcggtgg atctgtgaaa acacgggcat gtggacccgtgacggtctga cgtttttcag 60780 cgcccggggt gatgaaattc cgccgccgcg ctccatcatgttccatatct ggacggcgta 60840 cagtccgttc accacctggg tacagattgt ctatgactggctggatgcac tgaaagatcc 60900 caacggcctg aaaacctttg tgaacaccac gctgggcgagacctgggaag aggccgtggg 60960 cgaaaaactc gatcaccagg tactgatgga taaggtggtgcgttacacgg cggcggtgcc 61020 tgcccgggtg gtttatctga cggcgggcat tgactcgcagcgaaaccgtt ttgagatgta 61080 tgtctgggga tgggctccgg gagaggaagc ctttctggtggataaaatca tcattatggg 61140 gcgtcctgat gaggaagaga cgctgttacg tgtggatgcggcgatcaaca aaaaataccg 61200 ccatgcggat ggcaccgaaa tgactatttc ccgtgtctgctgggacaccg gggggatcga 61260 tggtgaaatt gtttatcaga gatcaaaaaa acacggtgttttccgggtgc tgccggtaaa 61320 aggcgcatct gtctatggca agccggtgat caccatgccaaaaacccgca atcagcgggg 61380 cgtgtatctg tgtgaagtgg gaacggacac cgcaaaagaaattctctatg cccgtatgaa 61440 agccgatccc tcgcctgcgg atgaagccac gtcgtatgccatccgttttc ctgatgatcc 61500 ggagattttt tcgcagacag aggcgcagca actggtggcggaagagctgg tggagaagtg 61560 ggaaaaagga aagatgcgtc tgctgtggga taacaaaaagcggcgtaacg aagcgctgga 61620 ctgcctggtg tatgcctacg cggcattacg tgtgtccgtgcaacgctggc agcttgatct 61680 ggctgtactg gcaaaatccc gggaagaaga gacgacccggccaaccctga aagaactggc 61740 agcgaagctg tccggaggag tgaatggtta cagtcgctgaactgcaggcg ctgcgtcagg 61800 cgcgccttga tttattaacc ggtaaacggg tggtgtctgtccagaaagat ggtcgcagaa 61860 ttgaatatac ggcggcttct ctggatgagc ttaaccgggcgatcaatgat gcggagtcgg 61920 tactggggac aacccggcgt cgccgtcgtc cgctgggagtgaggttatga aacgaacgcc 61980 tgtcctgatt gatgtgaacg gcgttccgct tcgtgagagtctcagctaca acgggggcgg 62040 tgcaggattt ggcgggcaaa tggcggagtg gttgccaccggcgcagagtg ccgatgcggc 62100 cctgctgccc gcgttgcgtc tggggaatgc ccgggcagatgatctggtgc gcaataacgg 62160 aatagcggcc aatgcggtgg cactgcataa ggatcacattgtcgggcata tgtttcttat 62220 cagctaccgt ccgaactggc gctggctggg gatgcgggagaccgcagcaa aaagctttgt 62280 cgatgaggtg gaggcggcct ggtcggaata cgccgaagggatgtctggcg agatcgacgt 62340 ggaaggaaaa cgcacgttca cggaatttat ccgtgaaggtgtgggcgttc atgcgtttaa 62400 cggcgaaatc tttgtgcagc cggtctggga tacggaaaccacgcagttat tccgtacgcg 62460 ttttaaagcc gtgagtccga aacgggtgga cacgccaggacacggtatgg ggaaccgttt 62520 tctgcgggvc ggggtgragg tcgatcgata tggccgtgccgttgcgtacc atatctgtga 62580 ggatgatttt cctcgctccg ggagtggacg atgggaacggatcccgcgtg aacttcccac 62640 cgggcgtccg gccatgctgc atattttcga gccggtggaggacgggcaga cccgtggggc 62700 caaccagttt tacagcgtca tggaacggct gaagatgctcgattccctgc aggcaacaca 62760 gcttcagtcg gccattgtga aagccatgta tgcagcgacgattgaaagtg accttgatac 62820 cgaaaaggcc tttgaatata tcgccggtgc gccgcaggggcagaaggata atccgcttat 62880 taatattctg gagaagttct ccagctggta tgacacgaataacgtgacgc tgggtggtgt 62940 caaaattccg caccttttcc ccggggatga tctgaaactacagactgcgc aggattcaga 63000 caatggattt tcggcgcttg aacaggcgct gctgcggtatatcgccgccg gtcttggcgt 63060 ttcctacgaa cagttgtccc gtgattactc gaaggtcagttattcaagtg ccagggcctc 63120 tgccaatgag tcgtggcgct attttatggg gcggcgaaaatttattgcgg cccggctggc 63180 cacgcagatg ttttcctact ggctggaaga ggcacttcttcgggggatta tccgtccgcc 63240 acgggcgcgt tttgattttt atcaggcgcg atcagcctggtcacgggcag agtggattgg 63300 tgccggaaga atggccattg acgggctcaa ggaggttcaggaatcggtga tgcgcattga 63360 ggccggactg agcacgtatg agaaagagcc ggcgctgatgggcgaggatt atcaggacat 63420 tttccgccag caggtcaggg aatctgcaga gcggcaaaaagccggactct cacgtccggt 63480 gtggatagcg caggcgtatc agcagcagat agcggagagtcgcaggccgg aagaggagac 63540 aacaccccgt gagacgtaat ctttcacaca ttattgccgcagcattcaat gaaccgctgc 63600 ttcyggagcc cgcctatgcg cgggttttct tttgcgcgctcgggcgcgag atgggggcag 63660 caagtctttc ggtaccacag cagcaggtac agcttgatgctcccggaatg ctggctgraa 63720 cggacgagta catggccgga ggtaaacgac cggcccgtgtttaccgggtg gtgaacggta 63780 ttgctgtact gccggtgacc ggcacgctgg tgcaccggctggggggtatg cggccatttt 63840 ccggaatgac aggctatgac ggcattgtcg cctgtcttcagcaggcaatg gcggatagcc 63900 aggtgcgggg cgtactgctg gacattgaca gtccgggcgggcaggccgcc ggcgcgtttg 63960 actgcgctga catgatttac cgcctccgtc agcagaagccggtctgggca ctgtgcaatg 64020 acacggcctg ttctgcagcc atgctgctgg cgtcggcctgctcccgacgg ctggttaccc 64080 agacatcccg tatcggctcc attggcgtga tgatgagccatgtcagctat gccggtcatc 64140 tggcgcaggc cggtgtggat atcacgctga tttactcaggggcgcacaag gtggatggca 64200 atcagtttga agccttaccg gcagaggttc gccagaacatgcagcagcgc attgatgcgg 64260 cgcgccggat gtttgccgaa aaagtggcca tgtttaccggtctgtctgtt gatgccgtca 64320 cgggaacaga ggccgccgtt tttgaaggtc agtccggcattgatgccggg ctggcggatg 64380 aattagtcaa tgcgtcggat gccatcagtg tgatggccacggcgctgaac agtaatgtca 64440 gaggaggcac tatgccgcaa ttaactgcaa cggaagccgccgcgcaggag aaccagcgag 64500 tgatggggat cctgacatgc caggaagcga aaggacgtgaacagcttgcc acgatgctgg 64560 caggacaaca gggcatgagc gttgaacagg cccgggcgattctggccgcg gcggcaccgc 64620 agcagccggt ggcatccacg cagagtgaag ccgatcgcattatggcgtgt gaagaagcga 64680 acggtcgtga acaactggcg gcaacgctgg cggcgatgccggagatgacg gtggaaaaag 64740 cccgcccgat cctggctgct tcaccgcagg cggatgccggaccctcactc cgtgatcaga 64800 tcatggcact ggatgaggca aaaggggctg aggcgcaggctgaacagctg gctgcctgcc 64860 cgggaatgac tgtggagagc gcccgggctg tgctggctgcgggatcaggt aaggcagaac 64920 cggtctctgc atccacaacc gccctgtttg aacgcatcatggcgaaccat tcaccggctg 64980 cggtacaggg tggcgtgcca cagacgtcag cagacggtgatgcggacgtg aaaatgctca 65040 tggccatgcc atgaagtcag tgctgaccat caacaggaggtttttacaat atggtaacga 65100 aaaacatcac tgaacagcgt gcggaagtac gtatttttgccggtaatgat ccggctcata 65160 ccgccacagg cagcagcggk atttcctcgg caacaccggcactgacgccc ctgatgctgg 65220 atgaagccag cgggaaactg gtggtctggg acggacagaaagccggtagt gcagttggca 65280 tactggtact gccgcttgaa ggcacagaga cggtactgacctattacaag tcggggacct 65340 ttgcgacgga ggcaatccgc tggcctgaaa gtgtggatgaacacaaaaag gcaaatgcct 65400 ttgccggcag tgccctgagt cacgcggcgc tgccgtaacacgttatcagg ccaccgcggt 65460 ggcctgactg atttctgaat gaaaggaact gatttatgggattgtttacg acccgccagt 65520 tactcggtta taccgaacaa aaagtgaaat ttcgtgcgctgtttctggag ctgtttttcc 65580 gccgtacggt gaatttccat accgaagagg tgatgctggacaaaattacc ggaaaaacgc 65640 cggtggcggc ctatgtctcc ccggttgttg aaggaaaagtgctgcgtcat cgcggtggtg 65700 aaacccgcgt gttacgtccg ggctacgtca agccgaaacacgaattcccc tggagccggt 65760 aaaaggagcc ggtaccaccc tgtgggttta taacggtcagggtgacgcct atgcaaaccc 65820 gttgtcagac gatgactggc agcgactggc taaggtgaaggatctgacgc cgggcgagat 65880 gacggcagaa tcctacgatg ataactacct ggatgatgaagacgcggact ggaccgcgac 65940 cgggcagggg cagaaatctg caggtgatac cagttttacgctggcctgga aaccgggaga 66000 ggaaggccag aaagggctta taggctggtt tgaaagcggcgatgtccggg cctataaaat 66060 ccgttttccg aatggcacgg tggatgtgtt tcgtggctgggtcagcagta tcggtaaggc 66120 cgtgacggcg aaagaagtga tcacccgcac ggtgaaagtcactaacgtgg gtaaaccttc 66180 tgtagcggaa gaacgcagca aaattacgcc ggtcagtgcgattaaggtga cgccgacatc 66240 cggtacggtg gcaaaaggga aaacaaccac cctgacggtttcttttgagc cggaaagtgc 66300 aacmgacaag acgttcagag cggtttccgc cgatccgtcgaaagccacca ttagtgtgaa 66360 agatatgaca attacggtaa acggcgtggc gacaggtaaggtgcagatcc ctgtggtgag 66420 cggaaatggt cagttcgccg cagtggctga agtcaccgttactgaagcgg gcgctgcagg 66480 gtaaacggag gtcatacatg tttctgaaaa cagaacaatttgaatataac ggtgtgtctg 66540 tcacgctttc cgaattgtct gcgctgcagc gtatcgagcatcttgccctc ctgaaacggc 66600 gtgcagaaca ggcagaatcc agcggcaacc tgcaggtaagcgtggaagat ctcgtcagaa 66660 ccggcgcgtt tctggtggcg atgtccctgt ggcataaccatccacagaaa acgcagtcac 66720 cgtcaatgaa tgaggccgtg atgaagatag agcaggaagtgctcaccacc tggcctgccg 66780 atgccattgc ccgggcggaa gacgtggtgt tgtgcctgtccgggatgagc ggggctgttc 66840 gtccggatac tgatattact gaagtggcga aaaataacacgctgactgat gatgattttt 66900 ctgcgggaaa gtcttcgacg gcgagctgaa ctttgccctcagactggcgc gtgagatggg 66960 gagacccgac tggcgcgcca tgcttgccgg gatgacatccaccgaatatg ccgactggca 67020 ccgtttttac cgcacgcatt attttcagga tacccagctggatatgcatt tttccgggct 67080 gacgtacgct gtactcagcc tgtttttttg cgatccggatatgcatccct ctgatttcag 67140 tctgcttgtc ccccggcatg aggaagagca ggtggagaggccggatgagg acaaaatgct 67200 gatgcagaaa gcggcaggac ttgccggagg cgtccggttcggtggggacg gagggcgcga 67260 tattttatcg tctgcggatg tggcggatgt catggtggatgatgccgcat taatgatggc 67320 ttcagcgggg attccgggag gtgtgagata tgtcccagccggttggtgat cttgttattg 67380 acctgagtct ggatgctgtc cgtttcgatg agcagatgagccgggtaagg cgtcattttt 67440 caggtctgga taccgacgtc agaaaaaccg ccagtgctgttgaacagggc ctgagccgcc 67500 aggcgctggc tgcacaaaaa gccgggattt ccgtcgggcagtataaagcg gccatgcgaa 67560 ccctgcccgc acagtttacg gatatcgcca cgcagcttgccggtggtcag aatccctggc 67620 tgatcctgct gcaacagggc ggtcaggtga aggactccttcggcgggatg atccccatgt 67680 tcagggggct tgccggtgcg atcaccctgc cgatggtcggggtcacctcg ctggcggtgg 67740 cgacaggtgc gctggtgtac gcctggtacc agggagattccacgctttca gcgtttaata 67800 aaaccctggt tctttccggt aatcagtccg gactgactgccgatcgcatg ctgacgctct 67860 caagagccgg gcaggcagca gggctgacgt ttaaccaggcgagagagtca ctggcagccc 67920 tggtgaatgc cggtgtgcgt ggtggtgaac agtttgatgccatcaaccag agtgtcgcgc 67980 gttttgcttc tgcatccggt gtggaggtgg acaaggttgcagaggctttc ggaaaactga 68040 ccaccgaccc tacgtcgggg ctgattgcga tggtgcgccagttccgtaac gtgacggcag 68100 agcagattgc gtatgttgcg cagctgcagc gttccggtgatgaggccggg gccttacagg 68160 cggcgaacga tatcgccacg aaaggctttg atgagcagacccgtcgcctg aaagaaaaca 68220 tggggacact ggagacctgg gcggataaaa ccgggaaggcattcaaatcg atgtgggatg 68280 ccattctgga tatcggtcgt cctgagtcct cagcggatatgctcgccagt gcacagaagg 68340 catttgatga ggcggataaa aaatggcagt ggtaccagagccggagccag cgccggggaa 68400 aaaccgcctc tttccgggcc aaccttcagg gcgcatggaatgaccgggaa aatgcccgtc 68460 tggggctggc agcggccacg ctgcagtcgg atatggaaaaagccggtgaa ctggccgcca 68520 gggaccgggc cgaacgggac gcatcacagc tgaagtataccggagaggcg cagaaggcgt 68580 atgagcgtct gctgacgccg ctggagaaat ataccgcccgtcaggaagaa ctgaataagg 68640 ccctgaaaga cgggaaaatc ctgcgggcgg attacaacacgctgatggcg gcggcgaaaa 68700 aggattatga atcgacgctg aaaaagccga agtcgtcaggagtcaaagtg tcagccggtg 68760 agcgtcagga agaccaggcg catgctgccc tgctggcgcttgaaaccgag ctcaggacgc 68820 tggaaaaaca cagcggtgcg aatgagaaaa tcagccagcagcgtcgcgat ttatggaaag 68880 cggaaaatca gtatgcggtc ctgaaagagg ctgccacgaaacggcagtta tctgagcagg 68940 aaaaattcct gctggcgcat aaagacgaga cgctggagtacaaacgccag ctggctgagc 69000 tgggagacaa agttgaacac cagaaacgcc tgaatgagctggcacagcag gcggtgcggt 69060 ttgaagagca gcagagcgcg aagcaggccg ccatcagcgcaaaagcccgc ggtctcactg 69120 accgtcaggc gcagcgggag tctgaagcgc agcgtcttcgggacgtgtac ggtgataatc 69180 cggctgcgct ggcgaaggcc acatctgcac tgaagaacacctggtctgcg gaggagcagc 69240 ttcgtggaag ctggatggcc gggctgaagt ccggctggggcgagtgggcg gaaagtgcga 69300 cggacagttt ttcgcaggtt aaaagtgctg ccacgcagacctttgacggt attgcacaga 69360 atatggcggc gatgctgacc ggtgcagagg cagactggcggggattcacc cgttcggtgc 69420 tgtccatgat gacagaaatc ctgcttaaac aggccatggtgggcattgtc gggcgtatcg 69480 gcagcgccat tggcggtgct ttcggtggtg gtgcatctgcttcctcgggg acggccattc 69540 aggctgcggc ggcgaacttc catttcgcga tcggaggatttacggggacg ggcggcaaat 69600 atgagcctgc gggaattgtt catcgcgggg agtttgttttcacgaaagag gcaaccagcc 69660 ggataggtgt ggggaatctt taccgtctga tgcgcggctatgcggaaggt ggttatgtgg 69720 gtggtgccgg aagtccggcg cagatgcggc gggcggaaggtattaatttt aatcagaaca 69780 atcacgtggt gattcagaac gacggcacca acggacaggcggggccgcag ctgatgaagg 69840 cggtgtatga catggcccgc aagggggcgc aggatgagctccggctgcag ttgcgtgatg 69900 gcggtatgtt atcggggagc gggcgatgaa aacctttcgctggaaagtga agccggatat 69960 ggaggtgaac tcgcagccat cggtgcgtga agtgcgttttggtgacgggt actcacagcg 70020 tatggcggca gggctgaatg ctgacctgaa aacataccgtgtgacgcttt ccgtgacccg 70080 ggaggaggcc cggcatctgg aagcgttcct ggcagagcacggaggctgga aggcattttt 70140 gtggaagcca ccctatgcat accggcagat aaaggtgacctgtgccgggt ggtctgcgcg 70200 ggtcgggatg ttgcgcgttg agttcagcgc ggagtttaagcaggtggtga actgatgcag 70260 gatattcacg aagaaagtct gaacgagtcg gttaaatcagagcagtcacc gcgggtggta 70320 ctctgggaaa tcgacctgac ggcgcagggc ggtgagcggtattttttctg caatgagctg 70380 aatgaaaaag gggaggcggt tacctggcag gggcggcaatatcaggcata cccgattgac 70440 ggcagtggct ttgagatgaa cgggaagggc agcagtgccagaccgtcgct gacggtgtcg 70500 aatctgtttg gtctggtcac cgggatggcg gaggacctgcagagcctggt gggggccacg 70560 gtggtccgcc gccgggtgta tgcccgtttt ctggatgcggtgaattttgt ggcaggcaay 70620 cctgaggcag accctgagca ggagctgacg gaccggtgggtggtggagca gatgtcatcg 70680 ctgacggcca tgacggcctc gtttgtgctg gcgacaccgacggagacgga cggagcgctg 70740 tttcccggtc gcattatgct ggcgaatacc tgtatgtgggattaccgggg agatgaatgc 70800 gggtataacg gtcctgcggt ggcggatgag ttcgacaaccccaccacgga tatccgtaag 70860 gacagatgca gcaagtgcat gcgcgggtgt gagatgcgcggcatggtggc taattttggc 70920 ggtttccttt ccatcaataa actttcgcag taaatcctgttttatgacac agactgaatc 70980 agcgattytg gtgcatgccc ggcggtgtgc gcctgcggagtcgtgcggct tcgtgatagg 71040 caccccggag ggcgaacggt accagccctg cgtgaatatctccgcagagc cggaggcgta 71100 ttttcgtatt gcgccggaag actggctgca ggcagagatgcagggggaga ttgtggcgct 71160 ggttcacagc caccctggtg gtctgccctg gctgagcgaggccgaccggc ggctgcagat 71220 aaagagtgcc ctgccctggt ggctggtctg ccggggggaaattcataaat tccgctgtgt 71280 gccgcacctg accgggcgtc gttttgagca cggggtgacggactgttaca ccctgttccg 71340 ggatgcatac catctggcgg ggataacgct gccggattttgagcgtgagg atgactggtg 71400 gcgcaacggt caraaccttt acctggacaa tatggcggcsactggttttt accgggtgcc 71460 cctgtcctct gcacaggcgg gcgatatcct gctgtgctgctttggcgcat cggtggccaa 71520 tcatgccgcc atatactgcg gcaacggtga actgcttcaccatctgcctg aacaactgag 71580 taaacgggag aggtattctg aaaaatggca acgacgaacgcattctgtct ggcgtcaccg 71640 ccactggcac gcatctgcct tcacggggat ttgcaacgatttggccgccg cctcagcctg 71700 tacgtgaaca cggcagcgga agccatccgt gccctgtcgctgcagatgcc gggattccgc 71760 cgtcagatga acgaaggctg gtaccagata cgtattcgcggtgaggacac ggcaccggag 71820 gcggtgtacg cccgtcttca cgaacctctg ggtgagggggcggtcatcca tattgtgccg 71880 cgactggccg gagccggaaa gggcggactg cagattgtgctgggggcagc agccatcgtg 71940 ggctctttct tcaccgcckg cgcaacgatg gcgttgtggggcgcagccct gagtgccgga 72000 gggctgactg ccaccacgat gctgttctca ctgggtgccagcatgatact gggcggtgtg 72060 gcccagatgc tggcaccgaa ggcaaaaaca ccggagtacagggcgacgga taacggtaaa 72120 cagaacacgt atttttcgtc actggataac atgattgcccaggggaaccc gatgccggtg 72180 ccttatggtg aaatgctggt tggttcacga cggatatcccaggacatcag cacccgtgat 72240 gagggcggag acgggaaagt ggtggttatc gggcggggatgaaaataaaa aaatcccgca 72300 gagttagcgg agctgcggga gagaacgatg aagattaacgttatggagtt atttttcagg 72360 catcaaaaaa gtaatgcagc gtcattattg cggctacaggcaattgccgg aaatgtgaag 72420 agtttcagaa attttattcc gtcatgacac aggcaccctccggggtgcct gttgttttct 72480 <210> SEQ ID NO 3 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 3 gatcttcttttttagagcgc cttg 24 <210> SEQ ID NO 4 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 4 tgcctgagttcacagataaa acac 24 <210> SEQ ID NO 5 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 5 gcgtaatgacttaatgattt tcgt 24 <210> SEQ ID NO 6 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 6 catcacattcctgacgcagt gctt 24 <210> SEQ ID NO 7 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 7 gagaatattatcagcgactt gata 24 <210> SEQ ID NO 8 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 8 ctagatcaactgagacagat tata 24 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 9 catgattggctggcgtccct 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:based on Escherichia coli <400> SEQUENCE: 10 accaatgaaa tgagttcaga 20<210> SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: based onEscherichia coli <400> SEQUENCE: 11 tgaaagtaaa cgaaaattgg cttc 24 <210>SEQ ID NO 12 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: based on Escherichiacoli <400> SEQUENCE: 12 aaagaatatc cggcccttct atct 24 <210> SEQ ID NO 13<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 13 atgttgagta tattgggcaa gaca 24 <210> SEQ ID NO 14 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 14 gaaatatcga taacagacgc tctc 24 <210> SEQ ID NO 15 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 15 gagaagcctt gcttcattaa agta 24 <210> SEQ ID NO 16 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 16 atgaagctgt tttggctgca ctat 24 <210> SEQ ID NO 17 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 17 atctgaaaga tctgcatttg atat 24 <210> SEQ ID NO 18 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 18 gattgtaagc taatatcagc tcat 24 <210> SEQ ID NO 19 <211>LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotides <400>SEQUENCE: 19 gaaacagcta tgaccatg 18 <210> SEQ ID NO 20 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic Nucleotides <400> SEQUENCE: 20 gtaaaacgacggccagt 17 <210> SEQ ID NO 21 <211> LENGTH: 23 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:based on Escherichia coli <400> SEQUENCE: 21 ataaatcgcc attcgttgac tac23 <210> SEQ ID NO 22 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: based onEscherichia coli <400> SEQUENCE: 22 gaacgcccac tgagatcat 19 <210> SEQ IDNO 23 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: based on Escherichiacoli <400> SEQUENCE: 23 ggcactgtct gaaactgctc c 21 <210> SEQ ID NO 24<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 24 tcgccagtta tctgacattc tg 22 <210> SEQ ID NO 25 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: based on Escherichia coli <400>SEQUENCE: 25 gacccggcac aagcataagc 20 <210> SEQ ID NO 26 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: based on Escherichia coli <400> SEQUENCE: 26ccacctgcag caacaagagg 20 <210> SEQ ID NO 27 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 27 gcatcatcaagcgtacgttc c 21 <210> SEQ ID NO 28 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 28 aatgagccaagctggttaag ct 22 <210> SEQ ID NO 29 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 29 gcaccattcatgatattcgt taac 24 <210> SEQ ID NO 30 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 30 ttgcaatgttcattaatata cgtc 24 <210> SEQ ID NO 31 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 31 tatactcattgataaaatac taac 24 <210> SEQ ID NO 32 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 32 agcacagaagagtaattata tgtc 24 <210> SEQ ID NO 33 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 33 atcaggatgccgttatactc attg 24 <210> SEQ ID NO 34 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 34 gcacagaagagtaattatat gtcc 24 <210> SEQ ID NO 35 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 35 aagttttgatattgtactgg atgc 24 <210> SEQ ID NO 36 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 36 cattaaagatagatgataaa tcac 24 <210> SEQ ID NO 37 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 37 tgctcaacatagaaacccac atag 24 <210> SEQ ID NO 38 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 38 tcgaatcagtgttatttacc agtg 24 <210> SEQ ID NO 39 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 39 gttattctggtacatgaaca tcat 24 <210> SEQ ID NO 40 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 40 tagataattccacacagccc acta 24 <210> SEQ ID NO 41 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 41 gtagtcgaaatcatggtgca gaat 24 <210> SEQ ID NO 42 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: based on Escherichia coli <400> SEQUENCE: 42 cttctctgctgtttggtgtc ttat 24

We claim:
 1. A method for typing the strain of a bacterial isolate, saidmethod comprising the steps of: (a) providing genomic DNA from abacterial isolate; (b) performing a polymerase chain reaction on thegenomic DNA using a first and second primer to amplify genomic DNAcomprising a restriction nuclease restriction site, thereby producing anamplicon having the restriction site; and (c) characterizing theamplicon of step (b), thereby typing the strain of the bacterialisolate.
 2. The method of claim 1, further comprising performing apolymerase chain reaction on genomic DNA of a reference strain of abacterial isolate using the first and second primers of step (b) toamplify genomic DNA of the reference strain of the bacterial isolate,and wherein step (c) is carried out by characterizing the amplicon ofthe reference strain of the bacterial isolate with the amplicon of step(b).
 3. The method of claim 1, wherein said amplicon of step (b)comprises at least 200-400 bp.
 4. The method of claim 1, wherein saidstrain of the bacterial isolate is a pathogenic strain.
 5. The method ofclaim 1, wherein said strain is a strain of E. coli O157:H7.
 6. Themethod of claim 2, wherein an amplicon is present in the bacterialisolate that is not present in said reference strain.
 7. The method ofclaim 2, wherein an amplicon is absent in said bacterial isolate that ispresent in said reference strain.
 8. The method of claim 2, whereinthere is an alteration in the size of said amplicon between saidbacterial isolate and said reference strain.
 9. The method of claim 2,wherein said reference strain of the bacterial isolate is E. coliO157:H7 strain 86-24.
 10. The method of claim 1, further comprisingdigesting the amplicon of step (b) with a restriction nuclease thatdigests the amplicon at the restriction site and where step (c) iscarried out by charactering the digestion products.
 11. The method ofclaim 10, wherein said digestion identifies a single nucleotidepolymorphism.
 12. The method of claim 10, wherein said single nucleotidepolymorphism identifies an additional site of restriction nucleasecleavage in said amplicon.
 13. The method of claim 10, wherein saidreference strain of the bacterial isolate is E. coli O157:H7 strain86-24.
 14. The method of claim 10, wherein said restriction site occursinfrequently in the genome of the bacterial isolate.
 15. The method ofclaim 10, wherein said restriction nuclease cleaves rarely within thegenome of the bacterial isolate.
 16. The method of claim 10, whereinsaid restriction nuclease is XbaI.
 17. The method of claim 10, whereinsaid restriction nuclease is AvrII.
 18. The method of claim 10, whereinsaid amplicon is digested with at least two restriction nucleases. 19.The method of claim 11, wherein said restriction nucleases are XbaI andAvrII.
 20. The method of claim 2, further comprising performing apolymerase chain reaction on genomic DNA of a reference strain of abacterial isolate using at least one pair of primers identifiedaccording to step (b) to amplify genomic DNA of the reference strain ofthe bacterial isolate and digesting said amplicon of the referencestrain with at least one restriction nuclease, and where step (c) iscarried out by characterizing the digestion products of the cleavedamplicon.
 21. A method for identifying a pair of primers for typing abacterial strain, said method comprising the steps of: (a) providinggenomic DNA of a bacterial strain; (b) fragmenting the genomic DNA ofthe bacterial strain into at least two fragments, wherein said fragmentsinclude a restriction enzyme site flanked by 5′ and 3′ regions of DNA;(c) identifying a first primer that hybridizes to the 5′ region flankingthe restriction site and a second primer that hybridizes to the 3′region flanking the restriction site, wherein said first and secondprimers amplify genomic DNA of the bacterial strain having therestriction site; (d) performing a polymerase chain reaction (PCR) onthe genomic DNA of the bacterial strain using the first and secondprimers of step (c) to amplify genomic DNA of the bacterial strain,thereby producing an amplicon; (e) providing a second genomic DNA, thesecond genomic DNA being from a reference bacterial strain, (f)performing a polymerase chain reaction (PCR) on the reference genomicDNA using the first and second primers of step (c) to amplify genomicDNA of the reference bacterial strain, thereby producing an amplicon;(i) comparing the amplicons of step (d) and step (f), wherein adifference between the amplicons of steps (d) and (f) identifies thepair of primers as a pair of primers for typing the bacterial strain.22. The method of claim 21, wherein said difference is the presence ofan amplicon not present in said reference strain.
 23. The method ofclaim 21, wherein said difference is the absence of an amplicon presentin said reference strain.
 24. The method of claim 21, wherein saiddifference is a difference in the size of said amplicons.
 25. The methodof claim 21, further comprising digesting the amplicon of step (d) andthe reference amplicon with a restriction nuclease that cleaves theamplicons at the restriction site, and detecting an alteration in thedigested amplicon of step (d) relative to the digested referenceamplicon, wherein a difference between the products of the digestedamplicons further identifies the pair of primers for typing thebacterial strain.
 26. The method of claim 25, wherein said digestionidentifies a single nucleotide polymorphism.
 27. The method of 25,wherein said single nucleotide polymorphism identifies an additionalsite of restriction endonuclease cleavage in said amplicon.
 28. Themethod of claim 25, wherein said restriction site occurs infrequently inthe genome of the bacterial strain.
 29. The method of claim 25, whereinsaid restriction nuclease cleaves rarely within the genome of thebacterial strain.
 30. The method of claim 25, wherein said restrictionnuclease is XbaI.
 31. The method of claim 25, wherein said restrictionnuclease is AvrII.
 32. The method of claim 25, wherein said amplicon isdigested with at least two restriction nucleases.
 33. The method ofclaim 32, wherein said restriction nucleases are XbaI and AvrII.
 34. Themethod of claim 25, wherein said amplicon of step (d) comprises at least200-400 bp.
 35. The method of claim 25, wherein said bacterial strain isa pathogenic strain.
 36. The method of claim 25, wherein said bacterialstrain is E. coli O157:H7.
 37. The method of claim 21, wherein thereference strain of step (e) includes a bacterial strain of E. coliO157:H7.
 38. The method of claim 37, wherein said reference strain is E.coli O157:H7 strain 86-24.
 39. A kit for distinguishing betweenbacterial strains comprising a set of primer pairs which, when used in aPCR reaction of genomic DNA from a sample of a bacterial isolate amplifyDNA across a site for a restriction endonuclease, the amplified DNAbeing polymorphic between strains of the bacteria.
 40. The kit of claim39, wherein said kit includes a set of primers identified according tothe method of claim
 13. 41. The kit of claim 39, said kit comprisingprimers for identifying a pathogenic bacterial strain.
 42. The kit ofclaim 39, wherein said strain is a strain of E. coli O157:H7.
 43. Abacterial strain typing profile, said typing profile produced accordingto the method of claim
 1. 44. The typing profile of claim 43, whereinsaid profile is depicted on an agarose gel.
 45. The typing profile ofclaim 43, wherein said profile is depicted on a dot blot.
 46. The typingprofile of claim 43, wherein said profile is depicted on a microarray.47. A microarray comprising at least two amplicons of a pathogenicbacterial strain.
 48. The microarray of claim 47, comprising acollection of amplicons.
 49. The microarray of claim 48, wherein saidcollection comprises at least five amplicons.
 50. The microarray ofclaim 48, wherein said collection comprises at least 10 amplicons. 51.The microarray of claim 48, wherein said collection comprises at least20 amplicons.
 52. The microarray of claim 47, wherein said amplicons, orfragments thereof, are produced according to the method of claim
 1. 53.The microarray of claim 47, wherein said pathogenic bacterial strainsare strains of E. coli O157:H7.
 54. A method for typing a strain of abacterial isolate, said method comprising the steps of: (a) providinggenomic DNA fragments from a bacterial isolate; (b) detectably labelingsaid fragments; (c) contacting the microarray of claim 44 with saiddetectably labeled fragments; and (c) determining the binding pattern ofsaid fragments to said microarray; thereby typing the strain of thebacterial isolate.
 55. The method of claim 54, wherein said bacterialstrain is a strain of E. coli O157:H7.
 56. The bacterial isolate ofclaim 54, wherein said isolate is from a patient.
 57. The bacterialisolate of claim 54, wherein said isolate is from a food source.
 58. Thebacterial isolate of claim 54, wherein said isolate is from soil. 59.The bacterial isolate of claim 54, wherein said isolate is from a watersource.
 60. A method of making a microarray, said method comprising thesteps of: (a) providing genomic DNA from at least one bacterial strain;(b) performing a polymerase chain reaction (PCR) on the genomic DNA ofthe bacterial strain using first and second primers to amplify genomicDNA of the bacterial strain, thereby producing an amplicon; and (c)affixing said amplicon to a solid support.
 61. The method of claim 60,wherein said polymorphic nucleic acid molecule is an amplicon, or afragment thereof.
 62. The method of claim 60, wherein said bacterialstrain is E. coli O157:H7.
 63. A method for typing a strain of abacterial isolate, said method comprising the steps of: (a) providinggenomic DNA from a bacterial isolate; (b) performing a polymerase chainreaction on the genomic DNA using a first and second primer to amplifygenomic DNA comprising a restriction nuclease restriction site; and (c)assaying for the presence or absence of an amplicon of step (b), therebytyping the strain of the bacterial isolate.